Human bikunin

ABSTRACT

The instant invention provides for proteins, polypeptides, nucleic acid sequences, constructs, expression vectors, host cells, pharmaceutical compositions of, and methods for using human placental bikunin, serine protease inhibitor domains, and fragments thereof.

FIELD OF THE INVENTION

The compositions of the invention relate to the field of proteins whichinhibit serine protease activity. The invention also relates to thefield of nucleic acid constructs, vectors and host cells for producingserine protease inhibiting proteins, pharmaceutical compositionscontaining the protein, and methods for their use.

BACKGROUND OF THE INVENTION

Problem Addressed

Blood loss is a serious complication of major surgeries such as openheart surgery and other complicated procedures. Cardiac surgery patientsaccount for a significant proportion of transfused donor blood. Bloodtransfusion carries risks of disease transmission and adverse reactions.In addition, donor blood is expensive and demands often exceed supply.Pharmacological methods for reducing blood loss and the resultant needfor transfusion have been described (reviewed by Scott et al., Ann.Thorac. Surg. 50: 843-851,1990).

Protein Serine Protease Inhibitor

Aprotinin, a bovine serine protease inhibitor of the Kunitz family isthe active substance in the medicament Trasylol®. Aprotinin (Trasylol®)has been reported as being effective in reducing perioperative bloodloss (Royston et al., Lancet ii: 1289-1291, 1987; Dietrich et al.,Thorac. Cardiovasc. Surg. 37: 92-98, 1989; Fraedrich et al., Thorac.Cardiovasc, Surg. 37: 89-91, 1989); W. van Oeveren et al. (1987), AnnThorac. Surg. 44, pp 640-645; Bistrup et al., (1988) Lancet 1, 366-367),but adverse effects, including hypotension and flushing (Bohrer et al.,Anesthesia 45: 853-854, 1990) and allergic reactions (Dietrich et al.,Supra) have been reported. Use of aprotinin in patients previouslyexposed to it is not recommended (Dietrich et al., Supra). Trasylol® hasalso been used for the treatment of hyperfibrinolytic hemorrhages andtraumatic hemorrhagic shock.

Aprotinin is known to inhibit several serine proteases includingtrypsin, chymotrypsin, plasmin and kallikrein, and is usedtherapeutically in the treatment of acute pancreatitis, various statesof shock syndrome, hyperfibrinolytic hemorrhage and myocardialinfarction (Trapnell et al., (1974) Brit J. Surg. 61: 177; J. McMichanet al., (1982) Circulatory Shock 9: 107; Auer et al, (1979) ActaNeurochir. 49: 207; Sher (1977) Am J. Obstet. Gynecol. 129: 164;Schneider (1976), Artzneim.-Firsch. 26: 1606). It is generally thoughtthat Trasylol® reduces blood loss in vivo through inhibition ofkallikrein and plasmin. It has been found that aprotinin (3-58, Arg15,Ala17, Ser42) exhibits improved plasma kallikrein inhibitory potency ascompared to native aprotinin itself (WO 89/10374).

Problems with Aprotinin

Because aprotinin is of bovine origin, there is a finite risk ofinducing anaphylaxis in human patients upon re-exposure to the drug.Thus, a human functional equivalent to aprotinin, by virtue of a lowerrisk of anaphylaxis, would be most useful and desirable to have.

Aprotinin is also nephrotoxic in rodents and dogs when administeredrepeatedly at high dose (Bayer, Trasylol®, Inhibitor of proteinase;Glasser et al., in “Verhandlungen der Deutschen Gesellschaft fur InnereMedizin, 78. Kongress”, Bergmann, Munchen, 1972 pp. 1612-1614). Onehypothesis ascribes this effect to the accumulation of aprotinin in thenegatively charged proximal tubules of the kidney, due to its high netpositive charge (WO 93/14120).

Accordingly, an object of the present invention is to identify humanproteins with functional activity similar to aprotinin. It was also anobject of the instant invention to identify human proteins, that wouldbe less charged, yet exhibit the same, highly similar, or improvedprotease specificities as found for aprotinin, especially with respectto the potency of plasmin and kallikrein inhibition. Such inhibitorscould then be used repeatedly as medicaments in human patients withreduced risk of adverse immune response and reduced nephrotoxicity.

BRIEF SUMMARY OF THE INVENTION

The instant invention provides for a purified human serine proteaseinhibitor which can specifically inhibit kallikrein, that has beenisolated from human placental tissue via affinity chromatography.

The instant invention provides a newly identified human protein hereincalled human placental bikunin that contains two serine proteaseinhibitor domains of the Kunitz class. In one particular embodiment, theinstant invention embodies a protein having the amino acid sequence:(SEQ ID NO: 1) ADRERSIHDF CLVSKVVGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100 NYEEYCTANAVTGPCRASFP RWYFDVERNS CNNFIYGGCR GNDNSYRSEE 150 ACMLRCFRQQ ENPPLPLGSKVWLAGAVS 170

In a prefered embodiment the instant invention provides for native humanplacental bikunin protein having the amino acid sequence: (SEQ ID NO:52) ADRERSIHDF CLVSKVVGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50 YLTKEECLKKCATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100 NYEEYCTANA VTGPCRASFPRWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150 ACMLRCFRQQ ENPPLPLGSK 170

In one aspect, the biological activity of the protein of the instantinvention is that it can bind to and substantially inhibit thebiological activity of trypsin, human plasma and tissue kallikreins,human plasmin and Factor XIIa. In a preferred embodiment, the presentinvention provides for a native human placental bikunin protein, inglycosylated form. In a further embodiment the instant inventionencompasses native human bikunin protein which has been formed such thatit contains at least one cysteine-cysteine disulfide bond. In apreferred embodiment, the protein contains at least one intra-chaincysteine-cysteine disulfide bond formed between a pair of cysteinesselected from the group consisting of CYS11-CYS61, CYS20-CYS44,CYS36-CYS57, CYS106-CYS156, CYS115-CYS139, and CYS131-CYS 152, whereinthe cysteines are numbered according to the amino acid sequence ofnative human placental bikunin. One of ordinary skill will recognizethat the protein of the instant invention may fold into the properthree-dimensional conformation, such that the biological activity ofnative human bikunin is maintained, where none, one or more, or all ofthe native intra-chain cysteine-cysteine disulfide bonds are present. Ina most preferred embodiment, the protein of the instant invention isproperly folded and is formed with all of the proper nativecysteine-cysteine disulfide bonds.

Active protein of the instant invention can be obtained by purificationfrom human tissue, such as placenta, or via synthetic protein chemistrytechniques, as illustrated by the Examples below. It is also understoodthat the protein of the instant invention may be obtained usingmolecular biology techniques, where self-replicating vectors are capableof expressing the protein of the instant invention from transformedcells. Such protein can be made as non-secreted, or secreted forms fromtransformed cells. In order to facilitate secretion from transformedcells, to enhance the functional stability of the translated protein, orto aid folding of the bikunin protein, certain signal peptide sequencesmay be added to the NH2-terminal portion of the native human bikuninprotein.

In one embodiment, the instant invention thus provides for the nativehuman bikunin protein with at least a portion of the native signalpeptide sequence intact. Thus one embodiment of the invention providesfor native human bikunin with at least part of the signal peptide,having the amino acid sequence: (SEQ ID NO: 2) AGSFLAWLGSLLLSGVLA −1ADRERSIHDFCLVSKVVGRCRASMPRWWYNVTDGSCQLFVYGGCDGNSNN 50YLTKEECLKKCATVTENATGDLATSRNAADSSVPSAPRRQDSEDHSSDMF 100NYEEYCTANAVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNSYRSEE 150ACMLRCFRQQENPPLPLGSKVVVLAGAVS 179

In a prefered embodiment the instant invention provides for a nativehuman placental bikunin protein with part of the leader sequence intact,having the amino acid sequence of SEQ ID NO: 52 with an intact leadersegment having the amino acid sequence: (SEQ ID NO: 53) MAQLCGLRRSRAFLALL GSLLLSGVLA −1

In another embodiment, the instant invention provides for bikuninprotein with part of the leader sequence intact, having the amino acidsequence of SEQ ID NO: 52 with the intact leader segment having theamino acid sequence: (SEQ ID NO: 54) MLR AEADGVSRLL GSLLLSGVLA −1

In a preferred numbering system used herein the amino acid numbered +1is assigned to the NH2-terminus of the amino acid sequence for nativehuman placental bikunin. One will readily recognize that functionalprotein fragments can be derived from native human placental bikunin,which will maintain at least part of the biological activity of nativehuman placental bikunin, and act as serine protease inhibitors.

In one embodiment, the protein of the instant invention comprises afragment of native human placental bikunin, which contains at least onefunctional Kunitz-like domain, having the amino acid sequence of nativehuman placental bikunin amino acids 7-159, hereinafter called “bikunin(7-0.159)”. Thus the instant invention embodies a protein having theamino acid sequence: (SEQ ID NO: 3)IHDFCLVSKVVGRCRASMPRWWYNVTDGSGQLFVYGGCDGNSNN 50YLTKEECLKKCATVTENATGDLATSRNAADSSVPSAPRRQDSEDHSSDMF 100NYEEYCTANAVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNSYRSEE 150 ACMLRCFRQ 159

where the amino acid numbering corresponds to that of the amino acidsequence of native human placental bikunin. Another functional variantof this embodiment can be the fragment of native human placentalbikunin, which contains at least one functional Kunitz-like domain,having the amino acid sequence of native human placental bikunin aminoacids 11-156, bikunin (11-156) (SEQ ID NO: 50)CLVSKWGRCRASMPRWWYNVTDGSCQLFVYGGCDGNSNN 50YLTKEECLKKCATVTENATGDLATSRNAADSSVPSAPRRQDSEDHSSDMF 100NYEEYCTANAVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNSYRSEE 150 ACMLRC. 156

One can recognize that the individual Kunitz-like domains are alsofragments of the native placental bikunin. In particular, the instantinvention provides for a protein having the amino acid sequence of afirst Kunitz-like domain consisting of the amino acid sequence of nativehuman placental bikunin amino acids 7-64, hereinafter called “bikunin(7-64)”. Thus in one embodiment the instant invention encompasses aprotein which contains at least one Kunitz-like domain having the aminoacid sequence: (SEQ ID NO: 4)IHDFCLVSKVVGRCRASMPRWWYNVTDGSCQLFVYGGCDGNSNN 50 YLTKEECLKKCATV 64

where the amino acid numbering corresponds to that of the amino acidsequence of native human placental bikunin. Another form of the proteinof the instant invention can be a first Kunitz-like domain consisting ofthe amino acid sequence of native human placental bikunin amino acids11-61, “bikunin (11-61)” having the amino acid sequence: (SEQ ID NO: 5)CLVSKVVGRGRASMPRWWYNVTDGSCQLFVYGGCDGNSNN 50 YLTKEECLKKC 61

The instant invention also provides for a protein having the amino acidsequence of a Kunitz-like domain consisting of the amino acid sequenceof native human placental bikunin amino acids 102-159, hereinaftercalled “bikunin (102-159)”. Thus one embodiment the instant inventionencompasses a protein which contains at least one Kunitz-like domainhaving the amino acid sequence: (SEQ ID NO: 6)YEEYCTANAVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNSYR 150 SEE ACMLRCFRQ 159

where the amino acid numbering corresponds to that of the amino acidsequence of native human placental bikunin. Another form of this domaincan be a Kunitz-like domain consisting of the amino acid sequence ofnative human placental bikunin amino acids 106-156, “bikunin (106-156)”having the amino acid sequence: (SEQ ID NO: 7)CTANAVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNSYRSEE 150 ACMLRC 156

Thus one of ordinary skill will recognize that fragments of the nativehuman bikunin protein can be made which will retain at least some of thenative protein biological activity—Such fragments can also be combinedin different orientations or multiple combinations to provide foralternative proteins which retain some of, the same, or more biologicalactivity of the native human bikunin protein.

One will readily recognize that biologically active protein of theinstant invention may comprise one or more of the instant Kunitz-likedomains in combination with additional Kunitz-like domains from othersources. Biologically active protein of the instant invention maycomprise one or more of the instant Kunitz-like domains in combinationwith additional protein domains from other sources with a variety ofbiological activities. The biological activity of the protein of theinstant invention can be combined with that of other known protein orproteins to provide for multifunctional fusion proteins havingpredictable biological activity. Thus, in one embodiment, the instantinvention encompasses a protein which contains at least one amino acidsequence segment the same as, or functionally equivalent to the aminoacid sequence of either SEQ ID NO: 5 or SEQ ID NO: 7.

An open reading frame which terminates at an early stop codon can stillcode for a functional protein. The instant invention encompasses suchalternative termination, and in one embodiment provides for a protein ofthe amino acid sequence: (SEQ ID NO: 8)ADRERSIHDFCLVSKVVGRCRASMPRWWUNVTDGSCQLFVYG 50 GCDGNSNNYLTKEECLKKCATVTENATGDLATSRNAADSSVPSAPRRQDS 92

In one embodiment, the instant invention provides for substantiallypurified, or recombinantly produced native human bikunin protein with anintact segment of the leader sequence, and at least a portion of thenative transmembrane region intact. Thus one embodiment of the inventionprovides for native human bikunin, with an intact leader sequence, andwith at least part of the transmembrane domain (underlined), having anamino acid sequence selected from: 1) EST                       MLRAEADGVSRLL GSLLLSGVLA −1 2) PCR                   MAQLCGL RRSRAFLALLGSLLLSGVLA −1 3) γcDNA                  MAQLCGL RRSRAFLALL GSLLLSGVLA−1 1) ADRERSIHDF CLVSKVVGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50 2)ADRERSIHDF CLVSKVVGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50 3) ADRERSIHDFCLVSKVVGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50 1) YLTKEECLKK CATVTENATGDLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100 2) YLTKEECLKK CATVTENATG DLATSRNAADSSVPSAPRRQ DSEDHSSDMF 100 3) YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQDSEDHSSDMF 100 1) NYEEYCTANA VTGPCRASFP RWYFDVERNS GNNFIYGGCR GNKNSYRSEE150 2) NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150 3)NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150 1) ACMLRCFRQQENPPLPLGSK VVVLAGLFVM VLILFLGASM VYLIRVARRN 200 2) ACMLRCFRQQ ENPPLPLGSKVVVLAGLFVM VLILFLGASM VYLIRVARRN 200 3) ACMLRCFRQQ ENPPLPLGSK VVVLAGLFVMVLILFLGASM VYLIRVARRN 200 1) QERALRTVWS SGDDKEQLVK NTYVL 225 2)QERALRTVWS FGD 213 3) QERALRTVWS SGDDKEQLVK NTYVL 225where sequence 1) is EST derived consensus SEQ ID NO: 45, 2) is PCRclone SEQ ID NO:47, and 3) is lambda cDNA clone SEQ ID NO:49. In apreferred embodiment a protein of the instant invention comprises one ofthe amino acid sequence of SEQ ID NO: 45, 47 or 49 wherein the proteinhas been cleaved in the region between the end of the last Kunitz domainand the transmembrane region.

The instant invention also embodies the protein wherein the signalpeptide is deleted. Thus the instant invention provides for a proteinhaving the amino acid sequence of SEQ ID NO: 52 continuous with atransmembrane amino acid sequence: (SEQ ID NO: 69) EST VVVLAGLFVMVLILFLGASM VYLIRVARRN 200 EST QERALRTVWS SGDDKEQLVK NTYVL 225

a transmembrane amino acid sequence: (SEQ ID NO: 68) PCR VVVLAGLFVMVLILFLGASM VYLIRVARRN 200 PCR QERALRTVWS FGD 213

or a transmembrane amino acid sequence: (SEQ ID NO: 67) γcDNA VVVLAGLFVMVLILFLGASM VYLTRVARRN 200 γcDNA QERALRTVWS SGDDKEQLVK NTYVL 225

The protein amino acid sequences of the instant invention clearly teachone of the art the appropriate nucleic acid sequences which can be usedin molecular biology techniques to produce the proteins of the instantinvention. Thus, one embodiment of the instant invention provides for anucleic acid sequence which encodes for a human bikunin having theconsensus DNA sequence of FIG. 3 (SEQ ID NO: 9), which translates intothe amino acid sequence for native human placental bikunin sequence ofFIG. 3 (SEQ ID NO: 10). In another embodiment, the instant inventionprovides for a consensus nucleic acid sequence of FIG. 4C (SEQ ID NO:51) which encodes for an amino acid sequence of FIG. 4D (SEQ ID NO: 45).

In a preferred embodiment, the instant invention provides for a nucleicacid sequence which encodes for native human placental bikunin havingthe DNA sequence of FIG. 4F (SEQ ID NO: 48) which encodes for theprotein sequence of SEQ ID NO: 49. In an another embodiment, the instantinvention provides for a nucleic acid sequence of FIG. 4E (SEQ ID NO:46) which encodes for a protein sequence of SEQ ID NO: 47.

One can easily recognize that certain allelic mutations, andconservative substitutions made in the nucleic acid sequence can be madewhich will still result in a protein amino acid sequence encompassed bythe instant invention. One of skill in the art can recognize thatcertain natural allelic mutations of the protein of the instantinvention, and conservative substitutions of amino acids in the proteinof the instant invention will not significantly alter the biologicalactivity of the protein, and are encompassed by the instant invention.

The instant invention also provides for pharmaceutical compositionscontaining human placental bikunin and fragments thereof that are usefulfor the reduction of perioperative blood loss in a patient undergoingsurgery.

The present invention also provides methods for reducing perioperativeblood loss in a patient undergoing surgery, wherein an effective amountof the disclosed human serine protease inhibitors of the presentinvention in a biologically compatible vehicle is administered to thepatient.

The present invention also provides for variants of placental bikunin,and the specific Kunitz domains described above, that contain amino acidsubstitutions that alter the protease specificity. Preferred sites ofsubstitution are indicated below as positions Xaa¹ through Xaa³² in theamino acid sequence for native placental bikunin. Substitutions at Xaa¹through Xaa¹⁶ are also preferred for variants of bikunin (7-64), whilesubstitutions at through Xaa¹⁷ through Xaa³² are preferred for variantsof bikunin (102-159).

Thus the present invention embodies protein having an amino acidsequence: (SEQ ID NO: 11) Ala Asp Arg Glu Arg Ser Ile Xaa¹ Asp Phe 10Cys Leu Val Ser Lys Val Xaa² Gly Xaa³ Cys 20 Xaa⁴ Xaa⁵ Xaa⁶ Xaa⁷ Xaa⁸Xaa⁹ Trp Trp Tyr Asn 30 Val Thr Asp Gly Ser Cys Gin Leu Phe Xaa¹⁰ 40 TyrXaa¹¹ Gly Cys Xaa¹² Xaa¹³ Xaa¹⁴ Ser Asn Asn 50 Tyr Xaa¹⁵ Thr Lys Glu GluCys Leu Lys Lys 60 Cys Ala Thr Xaa¹⁶ Thr Glu Asn Ala Thr Gly 70 Asp LeuSer Thr Ser Arg Asn Ala Ala Asp 80 Ser Ser Val Pro Ser Ala Pro Arg ArgGin 90 Asp Ser Glu His Asp Ser Ser Asp Met Phe 100 Asn Tyr Xaa¹⁷ Glu TyrCys Thr Ala Asn Ala 110 Val Xaa¹⁸ Gly Xaa¹⁹ Cys Xaa²⁰ Xaa²¹ Xaa²² Xaa²³Xaa²⁴ 120 Xaa²⁵ Trp Tyr Phe Asp Val Glu Arg Asn Ser 130 Cys Asn Asn PheXaa²⁶ Tyr Xaa²⁷ Gly Cys Xaa²⁸ 140 Xaa²⁹ Xaa³⁰ Lys Asn Ser Tyr Xaa³¹ SerGlu Glu 150 Ala Cys Met Leu Arg Cys Phe Arg Xaa³² Gin 160 Glu Asn ProPro Leu Pro Leu Gly Ser Lys 170 Val Val Val Leu Ala Gly Ala Val Ser. 179where Xaa¹-Xaa³² each independently represents a naturally occurringamino acid residue except Cys, with the proviso that at least one of theamino acid residues Xaa¹-Xaa³² is different from the corresponding aminoacid residue of the native sequence.

In the present context, the term “naturally occurring amino acidresidue” is intended to indicate any one of the 20 commonly occurringamino acids, i.e., Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu,Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val.

By substituting one or more amino acids in one or more of the positionsindicated above, it may be possible to change the inhibitor specificityprofile of native placental bikunin or that of the individualKunitz-like domains, bikunin (7-64) or bikunin (102-159) so that itpreferentially inhibits other serine proteases such as, but not limitedto, the enzymes of the complement cascade, TF/FVIIa, FXa, thrombin,neutrophil elastase, cathepsin G or proteinase-3.

Examples of preferred variants of placental bikunin include thosewherein Xaa¹ is an amino acid residue selected from the group consistingof His, Glu, Pro, Ala, Val or Lys, in particular wherein Xaa¹ is His orPro; or wherein Xaa² is an amino acid residue selected from the groupconsisting of Val, Thr, Asp, Pro, Arg, Tyr, Glu, Ala, Lys, in particularwherein Xaa² is Val or Thr; or wherein Xaa³ is an amino acid residueselected from the group consisting of Arg, Pro, Ile, Leu, Thr, inparticular wherein Xaa³ is Arg or Pro; or wherein Xaa⁴ is an amino acidresidue selected from the group consisting of Arg, Lys and Ser, Gin, inparticular wherein Xaa⁴ is Arg or Lys; or wherein Xaa⁵ is an amino acidresidue selected from the group consisting of Ala, Gly, Asp, Thr, inparticular wherein Xaa⁵ is Ala; or wherein Xaa⁶ is an amino acid residueselected from the group consisting of Ser, Lle, Tyr, Asn, Leu, Val, Arg,Phe, in particular wherein Xaa⁶ is Ser or Arg; or wherein Xaa⁷ is anamino acid residue selected from the group consisting of Met, Phe, Ile,Glu, Leu, Thr and Val, in particular wherein Xaa⁷ is Met or Ile; orwherein Xaa⁸ is an amino acid residue selected from the group consistingof Pro, Lys, Thr, Gin, Asn, Leu, Ser or Ile, in particular wherein Xaa⁸is Pro or He; or wherein Xaa⁹ is an amino acid residue selected from thegroup consisting of Arg, Lys or Leu, in particular wherein Xaa⁹ is Arg:or wherein Xaa¹⁰ is an amino acid residue selected from the groupconsisting of Val, Ile, Lys, Ala, Pro, Phe, Trp, Gin, Leu and Thr, inparticular wherein Xaa¹⁰ is Val; or wherein Xaa¹¹ is an amino acidresidue selected from the group consisting of Gly, Ser and Thr, inparticular wherein Xaa¹¹ is Gly; or wherein Xaa¹² is an amino acidresidue selected from the group consisting of Asp, Arg, Glu, Leu, Gin,Gly, in particular wherein Xaa¹² is Asp or Arg; or wherein Xaa¹³ is anamino acid residue selected from the group consisting of Gly and Ala; orwherein Xaa¹⁴ is an amino acid residue selected from the groupconsisting of Asn or Lys; or wherein Xaa¹⁵ is an amino acid residueselected from the group consisting of Gly, Asp, Leu, Arg, Glu, Thr, Tyr,Val, and Lys, in particular wherein Xaa¹⁵ is Leu or Lys; or whereinXaa¹⁶ is an amino acid residue selected from the group consisting ofVal, Gin, Asp, Gly, Ile, Ala, Met, and Val, in particular wherein Xaa¹⁶is Val or Ala; or wherein Xaa¹⁷ is an amino acid residue selected fromthe group consisting of His, Glu, Pro, Ala, Lys and Val, in particularwherein Xaa¹⁷ is Glu or Pro; or wherein Xaa¹⁸ is an amino acid residueselected from the group consisting of Val, Thr, Asp, Pro, Arg, Tyr, Glu,Ala or Lys, in particular wherein Xaa¹⁸ is Thr; or wherein Xaa¹⁹ is anamino acid residue selected from the group consisting of Arg, Pro, Ile,Leu or Thr, in particular wherein Xaa¹⁹ is Pro; or wherein Xaa²⁰ is anamino acid residue selected from the group consisting of Arg, Lys, Glnand Ser, in particular wherein Xaa²⁰ is Arg or Lys; or wherein Xaa²¹ isan amino acid residue selected from the group consisting of Ala, Asp,Thr or Gly; in particular wherein Xaa²¹ is Ala; or wherein Xaa²² is anamino acid residue selected from the group consisting of Ser, Ile, Tyr,Asn, Leu, Val, Arg or Phe, in particular wherein Xaa²² is Ser or Arg; orwherein Xaa²³ is an amino acid residue selected from the groupconsisting of Met, Phe, De, Glu, Leu, Thr and Val, in particular whereinXaa²³ is Phe or lie; or wherein Xaa²⁴ is an amino acid residue selectedfrom the group consisting of Pro, Lys, Thr, Asn, Leu, Gln, Ser or lie,in particular wherein Xaa²⁴ is Pro or Ile; or wherein Xaa²⁵ is an aminoacid residue selected from the group consisting of Arg, Lys or Leu, inparticular wherein Xaa²⁵ is Arg: or wherein Xaa²⁶ is an amino acidresidue selected from the group consisting of Val, He, Lys, Leu, Ala,Pro, Phe, Gln, Trp and Thr, in particular wherein Xaa²⁶ is Val or Ile;or wherein Xaa²⁷ is an amino acid residue selected from the groupconsisting of Gly, Ser and Thr, in particular wherein Xaa²⁷ is Gly; orwherein Xaa²⁸ is an amino acid residue selected from the groupconsisting of Asp, Arg, Glu, Leu, Gly or Gln, in particular whereinXaa²⁸ is Arg; or wherein Xaa²⁹ is an amino acid residue selected fromthe group consisting of Gly and Ala; or wherein Xaa³⁰ is an amino acidresidue selected from the group consisting of Asn or Lys; or whereinXaa³¹ is an amino acid residue selected from the group consisting ofGly, Asp, Leu, Arg, Glu, Thr, Tyr, Val, and Lys, in particular whereinXaa³¹ is Arg or Lys; or wherein Xaa³² is an amino acid residue selectedfrom the group consisting of Val, Gln, Asp, Gly, Ile, Ala, Met, and Thr,in particular wherein Xaa³² is Gin or Ala.

DESCRIPTION OF THE DRAWINGS

The invention will be better understood from a consideration of thefollowing detailed description and claims, taken in conjunction with thedrawings, in which:

FIG. 1 depicts the nucleotide sequence of EST R35464 (SEQ ID NO: 12) andthe translation of this DNA sequence (SEQ ID NO: 13) which yielded anopen reading frame with some sequence similarity to aprotinin. Thetranslation product contains 5 of the 6 cysteines in the correct spacingthat is characteristic for Kunitz-like inhibitor domains (indicated inbold). The position normally occupied by the remaining cysteine (atcodon 38) contained instead a phenylalanine (indicated by an asterisk).

FIG. 2 depicts the nucleotide sequence of EST R74593 (SEQ ID NO: 14),and the translation of this DNA sequence (SEQ ID NO: 15) which yieldedan open reading frame with homology to the Kunitz class of serineprotease inhibitor domains. The translation product contained 6cysteines in the correct spacing that is characteristic for Kunitz-likeinhibitor domains (indicated in bold). However, this reading framesequence includes stop codons at codon 3 and 23.

FIG. 3 depicts a deduced nucleic acid sequence of human placentalbikunin (SEQ ID NO: 9) labeled “consensus” and matched with thetranslated protein amino acid sequence labeled “translated” (SEQ ID NO:10). Also as comparison are shown the nucleic acid sequence for ESTsH94519 (SEQ ID NO: 16), N39798 (SEQ ID NO: 17), R74593 (SEQ ID NO: 14)and R35464 (SEQ ID NO: 12). The underlined nucleotides in the consensussequence correspond to the site of PCR primers described in theExamples. Underlined amino acids in the translated consensus sequenceare residues whose identity have been confirmed by amino acid sequencingof purified native human placental bikunin. Nucleotide and amino acidcode are standard single letter code, “N” in the nucleic acid codeindicates an unassigned nucleic acid, and “*” indicates a stop codon inthe amino acid sequence.

FIG. 4A depicts the original overlay of a series of ESTs with somenucleic acid sequence homology to ESTs encoding human placental bikunin,or portions thereof. Shown for reference are the relative positions ofbikunin (7-64) and bikunin (102-159), labeled KID1 and KID2respectively.

FIG. 4B depicts a subsequent more comprehensive EST overlayincorporating additional ESTs. Numbers on the upper X-axis refer tolength in base pairs, starting at the first base from the most 5′ ESTsequence. The length of each bar is in proportion to the length in basepairs of the individual ESTs including gaps. The EST accession numbersare indicated to the right of their respective EST bars.

FIG. 4C depicts the corresponding alignment of the oligonucleotidesequences of each of the overlapping ESTs shown schematically in FIG.4B. The upper sequence (SEQ ID NO: 51) labeled bikunin represents theconsensus oligonucleotide sequence derived from the overlappingnucleotides at each position. The numbers refer to base-pair positionwithin the EST map. The oligonucleotides in EST R74593 that are boldunderlined (at map positions 994 and 1005) are base insertions observedin R74593 that were consistently absent in each of the other overlappingESTs.

FIG. 4D depicts the amino acid translation of the consensusoligonucleotide sequence for bikunin depicted in FIG. 4C (SEQ ID NO:45).

FIG. 4E depicts the nucleotide sequence (SEQ ID NO: 46) andcorresponding amino acid translation (SEQ ID NO: 47) of a placentalbikunin encoding sequence that was derived from a human placental cDNAlibrary by PCR-based amplification.

FIG. 4F depicts the nucleotide sequence (SEQ ID NO: 48) andcorresponding amino acid translation (SEQ ID NO: 49) of a native humanplacental bikunin encoding clone that was isolated from a humanplacental lambda cDNA library by colony hybridization.

FIG. 4G compares the alignment of the amino acid translatedoligonucleotide sequences for placental bikunin obtained by EST overlay(SEQ ID NO: 45), PCR based cloning (SEQ ID NO: 47), and conventionallambda colony hybridization (SEQ ID NO: 49).

FIG. 5 shows a graph of purification of human placental bikunin fromplacental tissue after Superdex 75 Gel-Filtration. The plot is anoverlay of the protein elution profile as measured by OD 280 nm (solidline), activity of eluted protein in a trypsin inhibition assay (%inhibition shown by circles), and activity of eluted protein in akallikrein inhibition assay (% inhibition shown by squares).

FIG. 6 shows a graph which plots the purification of human placentalbikunin from placental tissue using C18 Reverse-Phase Chromatography.The plot is an overlay of the protein elution profile as measured by OD215 nm (solid line), activity of eluted protein in a trypsin inhibitionassay (% inhibition shown by circles), and activity of eluted protein ina kallikrein inhibition assay (% inhibition shown by squares).

FIG. 7 depicts a silver stained SDS-PAGE gel of highly purifiedplacental bikunin (lane 2), and a series of molecular size markerproteins (lane 1) of the indicated sizes in kilodaltons. Migration wasfrom top to bottom.

FIG. 8 shows the amount of trypsin inhibitory activity present in thecell-free fermentation broth from the growth of yeast strains SC101(panel 8A) or WHL341 (panel 8B) that were stably transformed with aplasmid (pS604) that directs the expression of placental bikunin(102-159).

FIG. 9 shows both a silver stained SDS-PAGE (left panel) and a Westernblot with anti-placental bikunin (102-159) pAb (right panel) ofcell-free fermentation broth from the growth of yeast strain SC101(recombinants 2.4 and 2.5) that was stably transformed with a plasmiddirecting the expression of either bovine aprotinin, or placentalbikunin (102-159). Migration was from top to bottom.

FIG. 10 is a photograph which shows a silver stained SDS-PAGE of highlypurified placental bikunin (102-159) (lane 2) and a series of molecularsize marker proteins (lane 1) of the indicated sizes in Kilodaltons.Migration was from top to bottom.

FIG. 11 is a photograph which shows the results of Northern blots ofmRNA from various human tissues that was hybridized to a ³²P labeledcDNA probe encoding either placental bikunin (102-159) (panel 11A) orencoding placental bikunin (1-213) (panel 11B). Migration was from topto bottom. The numbers to the right of each blot refer to the size inkilobases of the adjacent RNA markers. The organs from which mRNA wasderived is described under each lane of the blot.

FIG. 12 depicts an immunoblot of placental derived placental bikuninwith rabbit antiserum raised against either synthetic reduced placentalbikunin (7-64) (panel A) or 102-159 (panel B). For each panel, contentswere: molecular size markers (lanes 1); native placental bikuninisolated from human placenta (lanes 2); synthetic placental bikunin(7-64) (lanes 3) and synthetic placental bikunin (102-159) (lanes 4).Tricine 10-20% SDS-PAGE gels were blotted and developed with proteinA-purified primary polydonal antibody (8 ug IgG in 20 ml 0.1%BSA/Tris-buffered saline (pH 7.5), followed by alkalinephosphatase-conjugated goat anti-rabbit secondary antibody. Migrationwas from top to bottom.

FIG. 13 depicts a Coomassie Blue stained 10-20% Tricine SDS-PAGE gel of3 micrograms of highly purified placental bikunin (1-170) derived from abaculovirus/Sf9 expression system (lane 2). Lane 1 contains molecularsize markers. Migration was from top to bottom.

FIG. 14 depicts a comparison of the effect of increasing concentrationsof either Sf9-derived human placental bikunin (1-170) (filled circles),synthetic placental bikunin (102-159) (open circles), or aprotinin (opensquares) on the activated partial thromboplastin time of human plasma.Clotting was initiated with CaCl₂. The concentration of proteins areplotted versus the -fold prolongation in clotting time. The uninhibitedclotting time was 30.8 seconds.

DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses a newly identified human proteinherein called human placental bikunin that contains two serine proteaseinhibitor domains of the Kunitz class. The instant invention alsoencompasses pharmaceutical compositions containing placental bikunin andfragments thereof that are useful for the reduction of perioperativeblood loss in a patient undergoing surgery, or with major trauma.

The present invention also provides methods for reducing perioperativeblood loss in a patient undergoing surgery or due to major trauma,wherein an effective amount of the disclosed human serine proteaseinhibitors of the present invention, in a biologically compatiblevehicle, is administered to the patient.

A preferred application for placental bikunin, isolated domains, andother variants is for the reduction of blood loss resulting from traumaor surgery that has the potential for loss of large volumes of blood.These methods and compositions reduce or eliminate the need for wholedonor blood or blood products, thereby reducing the risk of infectionand other adverse side effects, as well as the cost of surgery. Themethods are thus useful in reducing blood loss in normal patients, i.e.,those not suffering from inborn or other pre-operative deficiencies incoagulation factors. The reduction in blood loss is seen as a reductionin blood loss during surgery, as reduced post surgical drainage or both.Preferred surgical applications include but are not limited to use inthoracic and abdominal surgery, total and partial hip replacementsurgeries and surgeries to treat a patient having an epithelial lesionof the eye. Preferred thoracic surgical procedures include but are notlimited to aortocoronary bypass, excision of cardiac and aorticaneurysms, and surgery for esophageal varices, and coronary arterybypass surgery. Preferred abdominal surgeries include but are notlimited to liver transplants, radical prostatectomy, surgery fordiverticulitis of colon, tumor debulking, surgery on the abdominal aortaand surgery for duodenal ulcers, and repair of liver or spleen trauma.Preferred use for the treatment of trauma include but are not limited tothe use in stabilization of severely injured patients at accident sitessuffering from e.g., limb loss or major thoracic/abdominal wounds. Incase of use for the reduction of blood loss resulting from surgery it ispreferred to administer the placental bikunin, isolated domains, orother variant prior to and during surgery, whereas in case of use intrauma settings the placental bikunin variant, isolated domain or othervariant is to be administered as rapidly as possible following injury,and should be contained on emergency vehicles traveling to the accidentsites.

Factor XII (also known as Hageman Factor) is a serine protease that isfound in the circulation in a zymogen form (80 kD) at approximately29-40 (μg/ml (see Pixley, et al. (1993) Meth. in Enz., 222, 51-64) andis activated by tissue and plasma kallikrein. Once activated, itparticipates in the intrinsic pathway of blood coagulation which isactivated when blood or plasma contacts a “foreign” or anionic surface.Once activated, Factor XIIa can then cleave and activate a number ofother plasma proteases including Factor XI, prekallikrein, and C1 of thecomplement system. Thus Factor XII may be involved in causinghypotensive reactions since activated kallikrein can cleave kininogenreleasing bradykinin (see Colman, (1984) J. Clin. Invest., 73,1249).

Sepsis is a disease that results from bacterial infection due tobacterial endotoxin or lipopolysaccharide (LPS). Exposure of Factor XIIto LPS results in the activation of Factor XII. Patients with sepsisfrequently have symptoms of intravascular coagulation which may also bedue to activation of Factor XII by LPS. Septic shock can result frombacterial infection and is associated with fever, low systemic vascularresistance, and low arterial pressure. It is a common cause of death inintensive care units in the United States, where seventy five percent ofthe patients that die from septic shock have a persistent hypotension(see Parillo, et al. (1989) Ann Rev. Med., 40,469-485).

Adult respiratory distress syndrome is characterized by pulmonary edema,hypoxemia, and decreased pulmonary compliance. The pathogenesis of thedisease is currently unknown although the proteolytic pathways ofcoagulation and fibrinolysis are believed to play a role (see Carvalho,et al. (1988) J. Lab Clin. Med., 112: 270-277).

The proteins of the instant invention are also a novel human Kunitz typeinhibitor of kallikrein, an activator of Factor XII. Thus another objectof the current invention is to present a method for the prophylactic ortherapeutic treatment of systemic inflammatory reactions such as septicshock, adult respiratory distress syndrome (ARDS), preeclampsia,multiple organ failure and disseminated intravascular coagulation (DIC).The therapeutic or prophylactic administration of the peptides of theinstant invention would result in the modulation of these inflammatoryconditions and be beneficial to the patient.

Plasmin plays an important role in extracellular matrix degradation andthe activation of matrix-metallo protease (MMP) cascades. Collectivelythese proteases mediate migration of and tissue invasion by bothendothelial cells during angiogenesis/neovascularization, and cancercells during metastasis. Neovascularization is essential to supporttumor growth and metastasis is a process which mediates the spreading oftumors and which is associated with extremely poor patient prognosis.

Several preclinical studies suggest that Kunitz like serine proteaseinhibitors with a protease specificity similar to aprotinin are usefulas medicaments for cancer. For example, aprotinin reduced tumor growthand invasion, with increased tumor necrosis when administered tohamsters bearing a highly invasive fibrosarcoma or to mice bearing asimilarly malignant mammary carcinoma (Latner et al., (1974), Br. J.Cancer 30: 60-67; Latner and Turner, (1976), Br. J. Cancer 33: 535-538).Furthermore, administration of 200,000 KIU of aprotinin i.p. to C57B1/6Cr male mice on days 1 to 14 post-inoculation with Lewis lung carcinomacells, reduced pulmonary metastases by 50% although had no effect onprimary tumor mass (Giraldi et al., (1977) Eur. J. Cancer, 13:1321-1323). Similarly, administration of 10,000 KIU i.p. on each of days13-16 post-inoculation of C57BL/6J mice with Lewis tumor cells inhibitedpulmonary metastases by 90% without affecting the primary tumor growth(Uetsuji et al., (1992), Jpn. J. Surg. 22: 429-442). In this same study,administration of plasmin or kallikrein with the same dosing schedulewas argued to increase the number of pulmonary metastases. These resultsprompted the authors to suggest that perioperative administration ofaprotinin to cancer patients may reduce the likelihood of metastases.Black and Steger (1976, Eur. J. Pharmacol., 38: 313-319) found thataprotinin inhibited the growth of the transplanted rodent Murphy-Strumlymphosarcoma in rats and suggested that the effect involved theinhibition of the kinin-forming enzyme system. Twice daily i.p.injection of female ddY mice with 10,000 KIU of aprotinin for 7 weeks tomice each bearing a single autochtonous squamous cell carcinomaresulting from 3-methylcholanthrene treatment reduced the growth rate ofthe primary tumors by 90%. In some animals rumor regression wasobserved. While all vehicle treated animals had died within the sevenweeks, all of the aprotinin treatment group remained alive. Reducedtumor growth was associated with hyperkeratosis (Ohkoshi, Gann (1980),71: 246-250).

Clinically, a surgically cured group of 26 patients who receivedaprotinin i.v. exhibited a 70% survival two years post surgery with norecurrence of tumors whereas a placebo group of 26 patients at the sametime exhibited only a 38% survival with a significant rate of tumorrecurrence (Freeman et al. Br. Soc. Gastroenterol. (1980) supplement A:902). In a case study (Guthrie et al., Br. J. Clin. Pract (1981) 35:330-332), administration of bromocriptine plus aprotinin to a patientwith advanced cancer of the cervix caused remission. Aprotinin wasadministerd both as a 500,000 KIU i.p. bolus every eight hoursconcurrently with a continuous i.v. infusion of aprotinin at a rate of200,000 KIU per 6 hr for a total of seven days once a month. Treatmentwas ended at the end of the fourth month due to the development of anallergic reaction to aprotinin. More recent evidence has furtherunderscored a role of plasmin as a target for these effects of aprotininon metastases.

The mechanism for these events could be related to the fact thataprotinin blocks the invasive potential of cancer cell lines (Liu G., etal., Int J. Cancer (1995), 60: 501-506). Furthermore, since the proteinsof the instant invention are also potent inhibitors of plasmin andkallikrien, they are contemplated for use as anti-cancer agents. Forexample they are contemplated for use in blocking primary tumor growthby restricting neovascularization, primary tumor invasion and inblocking metastasis through inhibition of tissue infiltration. Thecompounds may be administered locally to tumors or systemically. In apreferred mode of treatment, the protein would be administeredperioperatively during tumor debulking to minimize the risk ofmetastasis. In such a regime, the blood sparing properties of thecompound would be additionally advantageous in providing a clearersurgical field of view. Another preferred mode of administration wouldbe as a combination therapy with either MMP inhibitors or chemotherapy.An additional preferred mode of administration would be as a locallyadministered gene therapy designed to achieve selective expression ofplacental bikunin within the tumor cells, or their associated stroma andvascular beds.

Preferred types of cancers targeted for therapy would bevasular-dependent solid tumors such as breast, colon, lung, prostate andovarian carcinomas which exhibit a high metastatic potential, and thosefor which local delivery of a high concentration of the protein isfeasible such as lung cancers through pulmonary delivery, coloncarcinomas through hepatic delivery to liver metastasis, or skin cancerssuch as head and neck carcinomas or melanomas through subcutaneousdelivery. Since the proteins of the present invention are of humanorigin they would be less likely to be associated with allergic oranaphylactic reactions of the kind observed by Guthrie et al., supra,upon reuse.

Additionally, the proteins of the present invention are contemplated foruse in the reduction of thromboembolic complications associated withactivation of the intrinsic pathway of coagulation. This would includeprevention of pulmonary embolism in late stage cancer patients, afrequent cause of death (Donati MB., (1994), Haemostasis 24: 128-131).

Edema of the brain and spinal cord is a complication resulting fromtraumatic brain or spinal cord injury, stroke, cerebral ischemia,cerebral and sub-arachnoid hemhorrhage, surgery (including open heartsurgery), infectious diseases such as encephalitis and meningitis,granulomatous diseases such as Sarcoid and focal or diffuse carcinomas,and is a contributor to the high level of morbidity and death followingthese events. Bradykinin is known to disrupt the blood brain barrierexperimentally (Greenwood J., (1991), Neuroradiology, 33: 95-100;Whittle et al., (1992), Acta Neurochir., 115: 53-59), and infusion ofbradykinin into the internal carotid artery induced brain edema inspontaneously hypertensive rats (SHR) subjected to common carotid arteryocclusion (Kamiya, (1990), Nippon Ika Daigaku Zasshi. 57: 180-191).Elevated levels of bradykinin are found in extracellular fluidsfollowing trauma in a model involving traumatized rat spinal chord (Xuet al., (1991), J. Neurochem, 57: 975-980), and in plasma and tissuefrom rats with brain edema resulting from cerebral ischaemia (Kamiya etal., (1993), Stroke, 24: 571-575). Bradykinin is released from highmolecular weight kininogen by serine proteases including kallikrein(Coleman (1984) J. Clin Invest., 73: 1249), and the serine proteaseinhibitor aprotinin was found to block the magnitude of brain edemaresulting from cerebralschemia in SHR rats (Kamiya, (1990), Nippon IkaDaigaku Zasshi. 57: 180-191; Kamiya et al., (1993), Stroke, 24: 571-575)and rabbits subjected to a cold lesion of the brain (Unterberg et al.,(1986), J. Neurosurgery, 64: 269-276).

These observations indicate that brain edema results from localproteolytic release of kinins such as bradykinin from high molecularweight kininogen, followed by bradykinin-induced increases in bloodbrain barrier permeability. Accordingly, placental bikunin and fragmentsthereof are contemplated as medicaments for the prevention of edema inpatients at risk for this condition, particularly those of high risk ofmortality or brain injury. This would include head and spinal traumapatients, polytrauma patients, patients undergoing surgery of the brainor spinal cord and their associated vessels or other generalsurgeriesincluding open-heart surgery, patients who have suffered from a stroke,cerebral or sub-arachnoid hemorrhage, infectious diseases of the brain,granulomatous disease of the brain or diffuse or focal carcinomas andtumors of the brain or any conditions such as multiple sclerosisinvolving breakdown of the blood brain barrier or patients sufferingfrom any other inflammatory processes of the brain or spinal cord.Patients would receive an administration of placental bikunin either asan infusion or bolus injection, intravenously or intracranially.Additional doses of placental bikunin could be administeredintermittently over the following one to three weeks. Dose levels wouldbe designed to attain circulating concentrations in excess of thoserequired to neutralize elevations in plasma levels or bradykinin andother vasoactive peptides formed through the action of serine proteases,and sufficient to reduce edema. Since the protein is of human origin,repeated administration in this course of therapy would not lead todevelopment of an immune reaction to the protein. Placental bikunin andfragments thereof would be contemplated for monotherapy or prophylacsisas well as for use in combination with other medicaments such asneurotherapeutics and neuroprotectants.

Recent evidence (Dela Cadena R. A. at al., (1995), FASEB J. 9: 446-452)has indicated that the contact activation pathway may contribute to thepathogenesis of arthritis and anemia, and that kallikrein inhibitors maybe of therapeutic benefit. Accordingly, protease inhibitors of thepresent invention are contemplated according to their capacity toinhibit human kallikrein, as medicaments for the treatment of arthritisand anemia in humans.

Treatment of male non-insulin diabetic (NIDDM) patients with aprotininsignificantly improved total glucose uptake and decreased the metabolicclearance rate of insulin (Laurenti et al., (1996), Diabetic Medicine13: 642-645). Accordingly, the human proteins of the present inventionare contemplated for chronic use as medicaments for the treatment ofNIDDM.

Daily treatment of patients at risk of preterm delivery with urinarytrypsin inhibitor for two weeks significantly reduced recurrent uterinecontractions (Kanayama et al., (1996), Eur J. Obstet. Gynecol. & Reprod.Biol. 67: 133-138). Accordingly, the human proteins of the presentinvention are contemplated for use in the prevention of pretermdelivery.

Aprotinin has been shown to stimulate differentiation of mouse myoblastsin culture (Wells and Strickland, Development, (1994), 120: 3639-3647)),a process that is inhibited by TGFb. TGFb exists as an inactivepro-polypeptide which is activated by limited proteolysis. The mechanismof aprotinin action has been proposed to involve inhibition of proteaseswhich process pro-TGFb to the mature active form. TGFb has been shown tobe up-regulated in various fibrotic lesions and has long thought to be apotential target for anti-fibrotic therapies. In a rat model ofpulmonary fibrosis for example, TGF-b concentrations paralleled theextent of bleomycin-induced inflammation. Furthermore, plasmin levels inthe alveolar macrophage coincided with mature TGF-b levels, and theaddition of the plasmin inhibitor a-2-antiplasmin abrogated the posttranslational activation of pro-TGFb by the macrophage (Khal et al.,(1996), Am. J. Respir. Cell Mol. Biol. 15: 252-259.) The data suggestthat plasmin contributes to the formation of active TGFb by alveolarmacrophage, and that this process plays a pathologic role in thebleomycin-induced lung inflammation.

In light of these observations, placental bikunin and fragments thereofare contemplated as therapeutics for various fibrotic disorders,including pulmonary, hepatic, renal and dermal (scleroderma) fibrosis.

Aerosilized aprotinin was shown to protect >50% of mice infected withlethal doses of either influenza virus or paramyxovirus (Ovcharenko andZhimov, Antiviral Research, (1994), 23: 107-118). A suppression of thedevelopment of fatal hemorrhagic bronchopneumonia and a normalization ofbody weight gain were also noted with aerosilized aprotinin treatment.In light of these observations, placental bikunin and fragments thereofare contemplated as therapeutics for various respiratory relatedinfluenza-like diseases.

The human placental bikunin, isolated domains, and other variants of theinvention are contemplated for use in the medical/therapeuticapplications suggested for native aprotinin or aprotinin analogues withother inhibitory profiles, in particular those which necessitate usageof large doses. These would include diseases for which use of the humanprotein is indicated by virtue of its ability to inhibit human serineproteases such as trypsin, plasmin, kallikrein, elastase, cathepsin Gand proteinase-3, which include and are not limited to: acutepancreatitis (pancreatic elastase and trypsin), inflammation,thrombocytopenia, preservation of platelet function, organ preservation,wound healing, various forms of shock, including shock lung, endotoxinshock and post operative complications; disturbances of bloodcoagulation such as hyperfibrinolytic hemorrhage; acute and chronicinflammatory reactions, in particular for the therapy and prophylaxis oforgan lesions, such as for example pancreatitis and radiation inducedenteritis, complex-mediated inflammatory reactions such asimmunovasculitis, glomerulonephritis and types of arthritis;collagenoses in particular rheumatoid arthritis; types of arthritiscaused by metabolism-related deposits (for example gout); degenerationof the elastic constituents of the connective tissue parts of organs,such as in atherosclerosis (serum elastase) or pulmonary emphysema(neutrophil elastase); adult respiratory distress syndrome, inflammatorybowel disease, and psoriasis.

A major unexpected finding was that the synthetic peptides encodingbikunin (7-64), and bikunin (102-159), could properly fold into thecorrect three-dimensional conformation having active protease inhibitorbioactivity (Examples 2 and 1, respectively). Upon folding, each ofthese fragments of Bikunin underwent a reduction in mass of 6 massunits, consistent with the formation in each case, of three intrachaindisulfide bonds between six cysteine residues of each fragment. Anothersurprising finding is that the synthetic peptides encoding bikunin(7-64), bikunin (102-159), and bikunin (1-170) are highly inhibitory ofplasmin and both tissue and plasma kallikrein (Example 4, 3, and 10respectively). Inhibition of plasmin and kallikrein by Trasylol® isthought to be involved in the mechanism by which Trasylol® reduces bloodloss during open heart surgery. Our unexpected findings of thespecificity of the Kunitz domains of the present invention make themsuitable therapeutic agents for blood sparing during surgery or traumawhere there is significant blood loss, or for any other condition whereinhibition of plasmin and/or kallikrein would be beneficial.

Furthermore, we showed in this disclosure (Example 10) that placentalbikunin (1-170) is a potent inhibitor of factor XIa and a moderateinhibitor of factor Xa. Factor XIa plays an essential role in theintrinsic pathway of coagulation, serving to interconvert inactivefactor IX into active factor IXa. Thus, Placental Bikunin inhibits twokey enzymes of the intrinsic pathway, kallikrein and factor XIa.Consistent with these observations, we also showed that placentalbikunin (1-170) is a potent inhibitor of the activated partialthromboplastin time, which is a measure of the speed of coagulationdriven by the intrinsic pathway. On the other hand, we showed thatPlacental bikunin (1-170) is an extremely weak inhibitor of the tissuefactor VIIa complex, suggesting that it is not important in theregulation of the extrinsic coagulation cascade. Based on theseunexpected findings, placental bikunin is contemplated as a medicamentfor diseases in which activation of the intrinsic pathway of coagulationcontributes significantly to the disease mechanism. Examples of suchdiseases would include post-traumatic shock and disseminatedintravascular coagulation.

A significant advantage of the Kunitz domains of the present inventionis that they are human proteins, and also less positively charged thanTrasylol® (Example 1), thereby reducing the risk of kidney damage onadministration of large doses of the proteins. Being of human origin,the protein of the instant invention can thus be administered to humanpatients with significantly reduced risk of undesired immunologicalreactions as compared to administration of similar doses of Trasylol®.Furthermore, it was found that bikunin (102-159), bikunin (7-64), andbikunin (1-170) are significantly more potent inhibitors of plasmakallikrein than Trasylol® in vitro (Example 3, 4 and 10). Thus bikuninand fragments thereof are expected to be more effective in vivo atlowering blood loss in patients.

The amount of serine protease inhibitor administered should besufficient to provide a supra normal plasma level. For the prophylacticreduction of bleeding during and following coronary aortic by-passsurgery (CABG), the proteins of the instant invention may be used inplace of Trasylol® while taking into account the differences in potency.The use of Trasylol® is outlined in the Physicians Desk Reference,(1995), listing for Trasylol® supplement A. Briefly, with the patient ina supine position, the loading dose of placental bikunin, isolateddomain or other variant is given slowly over about 20 to 30 minutes,after induction of anesthesia but prior to sternotomy. In general, atotal dose of between about 2×10⁶ KIU (kallikrein inhibitory units) and8×10⁶ KIU will be used, depending on such factors as patient weight andthe length of the surgery. Preferred loading doses are those thatcontain a total of 1 to 2 million kallikrein inhibitory units (KIU).When the loading dose is complete, it is followed by the constantinfusion dose, which is continued until surgery is complete and thepatient leaves the operating room. Preferred constant infusion doses arein the range of about 250,000 to 500,000 KIU per hour. The pump primedose is added to the priming fluid of the cardiopulmonary bypasscircuit, by replacement of an aliquot of the priming fluid prior to theinstitution of the cardiopulmonary bypass. Preferred pump prime dosesare those that contain a total of about one to two million KIU.

The proteins of the instant invention are employed in pharmaceuticalcompositions formulated in the manner known to the art. Suchcompositions contain active ingredient(s) plus one or morepharmaceutically acceptable carriers, diluents, fillers, binders, andother excipients, depending on the administration mode and dosage formcontemplated. Examples of therapeutically inert inorganic or organiccarriers known to those skilled in the art include, but are not limitedto, lactose, corn starch or derivatives thereof, talc, vegetable oils,waxes, fats, polyols such as polyethylene glycol, water, saccharose,alcohols, glycerin and the like. Various preservatives, emulsifiers,dispersants, flavorants, wetting agents, antioxidants, sweeteners,colorants, stabilizers, salts, buffers and the like can also be added,as required to assist in the stabilization of the formulation or toassist in increasing bioavailability of the active ingredient(s) or toyield a formulation of acceptable flavor or odor in the case of oraldosing. The inhibitor employed in such compositions may be in the formof the original compound itself, or optionally, in the form of apharmaceutically acceptable salt. The proteins of the instant inventioncan be adminstered alone, or in various combinations, and in combinationwith other therapeutic compositions. The compositions so formulated areselected as needed for administration of the inhibitor by any suitablemode known to those skilled in the art.

Parenteral administration modes include intravenous (i.v.), subcutaneous(s.c), intraperitoneal (i.p.), and intramuscular (i.m.) routes.Intravenous administration can be used to obtain acute regulation ofpeak plasma concentrations of the drug as might be needed.Alternatively, the drug can be administered at a desired ratecontinuously by i.v. catheter. Suitable vehicles include sterile,non-pyrogenic aqueous diluents, such as sterile water for injection,sterile-buffered solutions or sterile saline. The resulting compositionis administered to the patient prior to and/or during surgery byintravenous injection or infusion.

Improved half-life and targeting of the drug to phagosomes such asneutrophils and macrophage involved in inflammation may be aided byentrapment of the drug in liposomes. It should be possible to improvethe selectivity of liposomal targeting by incorporating into the outsideof the liposomes ligands that bind to macromolecules specific to targetorgans/tissues such as the GI tract and lungs. Alternatively, i.m. ors.c. deposit injection with or without encapsulation of the drug intodegradable microspheres (e.g., comprising poly-DL-lactide-co-glycolide)or protective formulations containing collagen can be used to obtainprolonged sustained drug release. For improved convenience of the dosageform it is possible to use an i.p. implanted reservoir and septum suchas the percuseal system. Improved convenience and patient compliance mayalso be achieved by use of either injector pens (e.g., the Novo Pin orQ-pen) or needle-free jet injectors (e.g., from Bioject, Mediject orBecton Dickinson). Precisely controlled release can also be achievedusing implantable pumps with delivery to the desired site via a cannula.Examples include the subcutaneously implanted osmotic pumps availablefrom ALZA such as the ALZET osmotic pump.

Nasal delivery may be achieved by incorporating the drug intobioadhesive particulate carriers (<200 mm) such as those comprisingcellulose, polyacrylate or polycarbophil, in conjunction with suitableabsorption enhancers such as phospholipids or acylcarnitines.Commercially available systems include those developed by Dan Biosys andScios Nova.

Pulmonary delivery represents a nonparenteral mode of administration ofthe drug to the circulation. The lower airway epithelia are highlypermeable to a wide range of proteins of molecular sizes up to about 20kDa. Micron-sized dry powders containing the medicament in a suitablecarrier such as mannitol, sucrose or lactose may be delivered to thedistal alveolar surface using dry powder inhalers such as those ofInhale™, Dura™, Fisons (Spinhaler™), and Glaxo (Rotahaler™), or Astra(Turbohaler™) propellant based metered dose inhalers. Solutionformulations with or without liposomes may be delivered using ultrasonicnebulizers.

Oral delivery may be achieved by incorporating the drug into tablets,coated tablets, dragées, hard and soft gelatin capsules, solutions,emulsions, suspensions or enteric coated capsules designed to releasethe drug into the colon where digestive protease activity is low.Examples of the latter include the OROS-CT/Osmet™ system of ALZA, andthe PULSINCAP™ system of Scherer Drug Delivery Systems. Other systemsuse azo-crosslinked polymers that are degraded by colon-specificbacterial azoreductases, or pH sensitive polyacrylate polymers that areactivated by the rise in pH in the colon. The above systems may be usedin conjunction with a wide range of available absorption enhancers.Rectal delivery may be achieved by incorporating the drug intosuppositories.

In its preferred medicinal application, for reduction of perioperativeblood loss, the preferred mode of administration of the placentalbikunin variants of the present invention is parenterally, preferably byi.v. route through a central line.

The amount of the pharmaceutical composition to be employed will dependon the recipient and the condition being treated. The requisite amountmay be determined without undue experimentation by protocols known tothose skilled in the art. Alternatively, the requisite amount may becalculated, based on a determination of the amount of target proteasesuch as plasmin or kallikrein which must be inhibited in order to treatthe condition. As the active materials contemplated in this inventionare deemed to be nontoxic, treatment preferably involves administrationof an excess of the optimally required amount of active agent.

Additionally, placental bikunin, isolated domains or other variants maybe used to isolate natural substances such as its cognate proteases fromhuman material using affinity based separation methods, as well as toelicit antibodies to the protease that can be further used to explorethe tissue distribution and useful functions of Placental bikunin.

Searching Human Sequence Data

The existence of a distinct human protein homologous injunction toaprotinin, was deduced following a unique analysis of sequence entriesto the expressed-sequence-tag data-base (hereafter termed dbEST) at theNCBI (National Center for Biological Information, Maryland). Using theTBlastN algorithm (BLAST, or Basic Local Alignment Search Tool uses themethod of Altschul et al., (1990) J. Mol. Biol 215: 403-410, to searchfor similarities between a query sequence and all the sequences in adata-base, protein or nucleic acid in any combination), the data-basewas examined for nucleotide sequences bearing homology to the sequenceof bovine pre-pro-aprotinin, Trasylol®. This search of numerous cloneswas selectively narrowed to two particular clones which could possiblyencode for a deduced amino acid sequence that would correspond to ahuman protein homologous in function to aprotinin. The selected nucleicacid sequences were R35464 (SEQ ID NO: 12) and R74593 (SEQ ID NO: 14)that were generated from a human placental nucleic acid library. Thetranslated protein sequence in the longest open reading frame for R35464(SEQ ID NO: 13) was missing one of the 6 cysteines that are critical forformation of the Kunitz-domain covalent structure, meaning that thenucleic acid sequence of R35464 could not yield a functional inhibitor.Similarly, the longest translated open reading frame from clone R74593(SEQ ID NO: 15) contained a stop codon 5′ to the region encoding theKunitz like sequence, meaning that this sequence, could not betranslated to yield a functional secreted Kunitz domain. Thesignificance of these sequences alone was unclear. It was possible thatthey represented a) the products of pseudogenes, b) regions ofuntranslated mRNA, or c) the products of viable mRNA which had beensequenced incorrectly.

Discovery of Human Bikunin

To specifically isolate and determine the actual human sequence, cDNAprimers were designed to be capable of hybridizing to sequences located5′ and 3′ to the segment of cDNA encoding our proposed Kunitz likesequences found within R35464 and R74593. The primers used to amplify afragment encoding the Kunitz like sequence of R74593 were: (the 3′primerwith a HindIII site; SEQ ID NO:33) CGAAGCTTCATCTCCGAAGCTCCAGACG and (the5′primer with an XbaI site; SEQ ID NO:34)AGGATCTAGACAATAATTACCTGACCAAGGA.

These primers were used to amplify by PCR (30 cycles) a 500 base pairproduct from a human placental cDNA library from Clontech (MATCHMAKER,Cat #HL4003AB, Clontech Laboratories, Palo Alto, Calif.), which wassubcloned into Bluescript-SK+ and sequenced with the T3 primer with aSequenase™ kit version 2.0. Surprisingly, the sequence of the fragmentobtained using our primers was different from the sequence listed in thedbEST data base for clone R74593. In particular, our new sequencecontained an additional guanosine base inserted 3′ to the putative stopcodon, but 5′ to the segment encoding the Kunitz-like sequence (FIG. 3).The insertion of an additional G shifted the stop codon out of thereading frame for the Kunitz-like domain (G at base pair 114 of thecorrected sequence for R74593; FIG. 3).

Subsequent query of the dbEST for sequences homologous to theKunitz-like peptide sequence of R74593 yielded H94519 derived from humanretina library and N39798. These sequences contained a Kunitz-likesequence that was almost identical to the Kunitz-like domain encoded inR35464 except that it contained all six of the characteristic cysteines.Overlay of each of the nucleotide sequences with that of R74593(corrected by the insertion of G at b,p, 114) and R35464 was used toobtain a consensus nucleotide sequence for a partial human placentalbikunin (SEQ ID NO: 9; FIG. 3). The translated consensus sequenceyielded an open reading frame extending from residue −18 to +179 (FIG.3; full translation SEQ ID NO: 10) that contained two completeKunitz-like domain sequences, within the region of amino acid residues17-64 and 102-159 respectively.

Further efforts attempted to obtain additional 5′ sequence by queryingdbEST with the sequence of R35464. Possible matches from such searches,that possessed additional 5′ sequence were then in turn used to re-querythe dbEST. In such an iterative fashion, a series of overlapping 5′sequences were identified which included clones H16866, T66058, R34808,R87894, N40851 and N39876 (FIG. 4). Alignment of some of these sequencessuggested the presence of a 5′ ATG which might serve as a start site forsynthesis of the consensus translated protein sequence. From thisselected information, it was now possible to selectively screen for, anddetermine the nucleic acid and polypeptide sequences of a human proteinwith homologous function to aprotinin.

Re-interrogation of the dbEST revealed a number of new EST entries shownschematically in FIG. 4B. Overlap with these additional ESTs allowed usto construct a much longer consensus oligonucleotide sequence (FIG. 4C)that extended both 5′ and 3′ beyond the original oligonucleotidesequence depicted in FIG. 3. In fact, the new sequence of total length1.6 kilobases extended all the way to the 3′ poly-A tail. The increasednumber of overlapping ESTs at each base-pair position along the sequenceimproved the level of confidence in certain regions such as the sequenceoverlapping with the 3′ end of EST R74593 (FIG. 3). Several overlappingESTs in this region corroborated two critical base deletions relative toR74593 (located as bold underlined in FIG. 4C, map positions 994 and1005). Translation of the new consensus sequence (FIG. 4D) in thebikunin encoding frame yielded a form of placental bikunin that waslarger (248 amino acids) than the mature sequence (179 amino acids)encoded from the original consensus (SEQ ID NO: 1), and was terminatedby an in-frame stop codon within the oligonucleotide consensus. The sizeincrease was due to a frame shift in the 3′ coding region resulting fromremoval of the two base insertions unique to EST R74593. The frame shiftmoved the stop codon of the original consensus (FIG. 3) out of frameenabling read through into a new frame encoding the additional aminoacid sequence. The new translation product (FIG. 4D) was identical tothe original protein consensus sequence (SEQ ID NO: 1) between residues+1 to +175 (encoding the Kunitz domains), but contained a new C-terminalextension exhibiting a putative 24 residue long transmembrane domain(underlined in FIG. 4D) followed by a short 31 residue cytoplasmicdomain. The precise sequence around the initiator methionine and signalpeptide was somewhat tentative due to considerable heterogeneity amongstthe overlapping ESTs in this region.

Analysis of the protein sequence by Geneworks™, highlighted asparagineresidues at positions 30 and 67 as consensus sites for putative N-linkedglycosylation. Asparagine 30 was not observed during N-terminalsequencing of the full length protein isolated from human placenta,consistent with it being glycosylated.

Cloning of Human Bikunin

The existence of a human mRNA corresponding to the putative humanbikunin nucleotide sequence inferred from the analysis of FIG. 3, wasconfirmed as follows. The nucleic acid primer hybridizing 5′ to theKunitz-encoding cDNA sequence of R35464 (b.p. 3-27 of consensusnucleotide sequence in FIG. 3): GGTCTAGAGGCCGGGTCGTTTCTCGCCTGGCTGGGA (a5′ primer derived from R35464 sequence with an XbaI site; SEQ ID NO:35), and the nucleic acid primer hybridizing 3′ to the Kunitz encodingsequence of R74593 (b.p. 680-700 of consensus nucleotide sequence inFIG. 3), was used to PCR amplify, from a Clontech human placentallibrary, a fragment of the size (ca. 670 b.p) expected from a cDNAconsensus nucleotide sequence encoding the placental bikunin sequence ofFIG. 3 (Shown schematically in FIG. 4A).

Using a 5′ primer hybridizing to a sequence in R87894 that is 126 b.p 5′to the putative ATG start site discussed above, (shown schematically inFIG. 4A at b.p. 110) plus the same 3′ primer to R74593 as used above, itwas possible to amplify a fragment from a Clontech human placentallibrary of the expected size (approximately 872 b.p) predicted by ESToverlay (Shown schematically in FIG. 4).

Sequencing of the 872 b.p. fragment showed it to contain nucleotidesegment corresponding to b.p. 110 to 218 of EST R87894 at its 5′ end andb.p. 310 to 542 of the consensus sequence for placental bikunin inferredfrom the EST overlay analysis (of FIG. 3), at its 3′ end. This 3′nucleotide sequence contained all of the Kunitz-like domain encoded byplacental bikunin (102-159).

To obtain a cDNA encoding the entire extracellular region of theprotein, the following 5′ PCR primer: CACCTGATCGCGAGACCCC (SEQ ID NO:36)

designed to hybridize to a sequence within EST R34808 was used with thesame 3′ primer to EST 74593 to amplify (30 cycles) an approximately 780base-pair cDNA product from the human placental cDNA library. Thisproduct was gel purified, and cloned into the TA vector (Invitrogen) forDNA sequencing by the dideoxy method (Sanger F., et al., (1977) Proc.Natl. Acad. Sci (USA), 74: 5463-5467) with the following primers: VectorSpecific: GATTTAGGTGACACTATAG (SP6) (SEQ ID NO: 37) TAATACGACTCACTATAGGG(T7) (SEQ ID NO: 38) Gene Specific: TTACCTGACCAAGGAGGAGTGC (SEQ ID NO:39) AATCCGCTGCATTCCTGCTGGTG (SEQ ID NO: 40) CAGTCACTGGGCCTTGCCGT (SEQ IDNO: 41)

The resulting cDNA sequence is depicted in FIG. 4E together with itstranslation product. At the nucleotide level, the sequence exhibitedonly minor differences from the consensus EST sequence (FIG. 4D).Translation of the sequence yielded a coding sequence containing anin-frame initiator ATG site, signal peptide and mature placental bikuninsequence and transmembrane domain. The translated sequence of the PCRproduct was missing the last 12 amino acid residues from the cytoplasmicdomain as a consequence of the choice of selection of the 3′ primer forPCR amplification. This choice of 3′ PCR primer (designed based on thesequence of R74593) was also responsible for the introduction of anartifactual S to F mutation at amino acid position 211 of the translatedPCR-derived sequence. The signal peptide deduced from translation of thePCR fragment was somewhat different to that of the EST consensus.

To obtain a full length placental bikunin cDNA, the PCR derived product(FIG. 4E) was gel purified and used to isolate a non-PCR based fulllength clone representing the bikunin sequence. The PCR derived cDNAsequence was labeled with ³²P-CTP by High Prime (Boehringer Mannheim)and used to probe a placental cDNA Library (Stratagene, Unizap™ λlibrary) using colony hybridization techniques. Approximately 2×10⁶phage plaques underwent 3 rounds of screening and plaque purification.Two clones were deemed full length (˜1.5 kilobases) as determined byrestriction enzyme analysis and based on comparison with the size of theEST consensus sequence (see above). Sequencing of one of these clone bythe dideoxy method yielded the oligonucleotide sequence depicted in FIG.4F. The translation product from this sequence yielded a protein withinframe initiator methionine, signal peptide and mature placentalbikunin sequence. The mature placental bikunin sequence was identical tothe sequence of the mature protein derived by translation of the ESTconsensus although the signal peptide sequence lengths and sequencesdiffered. Unlike the PCR derived product, the cDNA derived by colonyhybridization contained the entire ectodomain, transmembrane domain,cytoplasmic domain and in-frame stop codon. In fact, the clone extendedall the way to the poly-A tail. The initiator methionine was followed bya hydrophobic signal peptide which was identical to the signal peptideencoded in the PCR derived clone. Subsequently we expressed and purifieda soluble fragment of placental bikunin, bikunin (1-170), from Sf9 cells(Example 9), and found it to be a functional protease inhibitor (Example10). Furthermore, we isolated from human placenta a soluble fragment ofplacental bikunin which was also an active protease inhibitor (Example7). Both the natural protein and the form of the protein expressed inSf9 cells are probably glycosylated at the asparagines residue atposition 30 based on the recoveries of PTH-amino acids during N-terminalsequencing (Examples 7 and 9).

Based on the above observations, it seems that full length placentalbikunin has the capacity to exist as a transmembrane protein on thesurface of cells as well as a soluble protein. Other transmembraneproteins that contain Kunitz domains are known to undergo proteolyticprocessing to yield mixtures of soluble and membrane associated forms.These include two forms of the Amyloid Precursor Protein termed APP751(Esch F., et al., (1990) Science, 248: 1122-1124) and APP 770 (Wang R.,et al., (1991), J. Biol Chem, 266:16960-16964).

Contact activation is a process which is activated by exposure ofdamaged vascular surfaces to components of the coagulation cascade.Angiogenesis is a process that involves local activation of plasmin atendothelial surfaces. The specificity of placental bikunin and itsputative capacity to anchor to cell surfaces, suggest that thephysiologic functions of transmembranous placental bikunin may includeregulation of contact activation and angiogenesis.

The amino acid sequences for placental bikunin (7-64), bikunin(102-159), and full length placental bikunin (FIG. 4F) were searchedagainst the PIR (Vers. 46.0) and PatchX (Vers. 46.0) protein databasesas well as the GeneSeq (Vers. 20.0) protein database of patentedsequences using the Genetics Computer Group program FastA. Using theGenetics Computer Group program TFastA (Pearson and Lipman, 1988, Proc.Natl. Acad. Sci. USA 85: 2444-2448), these same protein sequences weresearched versus the six-frame translations of the GenBank (Vers. 92.0with updates to Jan. 26, 1996) and EMBL (modified Vers. 45.0) nucleotidedatabases as well as the GeneSeq (Vers. 20.0) nucleotide database ofpatented sequences. The EST and STS subsets of GenBank and EMBL were notincluded in this set of searches. The best matches resulting from thesesearches contained sequences which were only about 50% identical overtheir full length to the 58-amino acid protein sequence derived from ouranalysis of clones R74593 and R35464.

Isolation of Human Bikunin

As mentioned above, synthetic peptides corresponding to bikunin (7-64)and bikunin (102-159) as determined from the translated consensussequence for bikunin (FIG. 3), could be refolded (Examples 2 and 1,respectively) to yield active kallikrein inhibitor protein (Example 4and 3, respectively). We exploited this unexpected property to devise apurification scheme to isolate native placental bikunin from humantissue.

Using a purification scheme which employed kallikrein-sepharose affinitychromatography as a first step, highly purified native potent kallikreininhibitor was isolated. The isolated native human bikunin had anidentical N-terminus (sequenced for 50 amino acid residues) as thesequence predicted by the translation of the consensus nucleic acidsequence (FIG. 3) amino acid residues +1 to +50 (Example 7). Thisconfirmed for the first time the existence of a novel native kallikreininhibitor isolated from human placenta.

Known Kunitz-like domains are listed below. Residues believed to bemaking contact with target proteases are highlighted as of specialinterest (bold/underlined). These particular residues are namedpositions Xaa¹⁻¹⁶ for specific reference as shown by label Xaa below:     Xaa                                1  1 111     1            1     1        2  3  456789              0  1 234     5            6  1)IHDFCLVSKVV GRCRASMPRW WYNVTDGSCQ LFVYGGCDGN SNNYLTKEEC LKKCATV  2)YEEYCTANAVT GPCRASFPRW YFDVERNSCN NFIYGGCRGN KNSYRSEEAC MLRCFRQ  3)-HSFCAFLADD GPCKAIMKRF FFNIFTRQCE EFIYGGCEGN QNRFESLEEC KKMCTRD  4)-PDFCFLEEDP GICRGYITRY FYNNQTKQCE RFKYGGCLGN MNNFETLEEC KNICEDG  5)-PSWCLTPADR GLCRANENRF YYNSVIGKCR PFKYSGCGGN ENNFTSKQEC LRACKKG  6)-AEICLLPLDY GPCRALLLRY YYRYRTQSCR QFLYGGCEGN ANNFYTWEAC DDACWRI  7)-PSFCYSPKDE GLCSANVTRY YFNPRYRTCD AFTYTGCGGN DNNFVSREDC KRACAKA  8)-KAVCSQEAMT GPCRAVMPRT TFDLSKGKCV RFITGGCGGN RNNEESEDYC MAVCKAM  9)RPDFCLEPPYT GPCKARIIRY FYNAKAGLCQ TFVYGGCRAK RNNFKSAEDC MRTCGGA 10)----CQLGYSA GPCMGMTSRY FYNGTSMACE TFQYGGCMGN GNNFVTEKEC LQTC 11) VAACNLPIVR GPCRAFIQLW AFDAVKGKCV LFPYGGCQGN GNKFYSEKEC REYCGVP 12)-EVCCSEQAET GPCRAMISRW YFDVTEGKCA PFFYGGCGGN RNNFDTEEYC MAVCGSA 13)----CKLPKDE GTCRDFILKW YYDPNTKSCA RFWYGGCGGN ENKFGSQKEC EKVC 14)-PNVCAFPMEK GPCQTYMTRW FFNFETGECE LFAYGGCGGN SNNFLEKEKC ECKCKFTwhere sequence number 1) is Bikunin (7-64) (SEQ ID NO: 4); sequence 2)is Bikunin (102-159) (SEQ ID NO: 6); sequence 3) is Tissue factorpathway inhibitor precursor 1 (SEQ ID NO: 18); sequence 4) is Tissuefactor pathway inhibitor precursor 1 (SEQ ID NO: 19); sequence 5) isTissue factor pathway inhibitor precursor (SEQ ID NO: 20); sequence 6)is Tissue factor pathway inhibitor precursor 2 (SEQ ID NO: 21); sequence7) is Tissue factor pathway inhibitor precursor 2 (SEQ ID NO: 22);sequence 8) is Amyloid precursor protein homologue (SEQ ID NO: 23);sequence 9) is Aprorinin (SEQ ID NO: 24); sequence 10) isInter-α-trypsin inhibitor precursor (SEQ ID NOs: 25); sequence 11) isInter-α-trypsin inhibitor precursor (SEQ ID NOs: 26); sequence 12) isAmyloid precursor protein (SEQ ID NO: 27); sequence 13) is Collagenα-3(VI) precursor (SEQ ID NO: 28); and squence 14) is HKI-B9 (SEQ ID NO:29).

It can be seen that Placental Bikunin (7-64) and (102-159) each have thesame number (six) and spacing of cysteine residues as is found inmembers of the Kunitz class of serine protease inhibitors. The precisebonding of cysteine residues to form the three intrachain disulfidebonds is known and invarient for all previously known Kunitz familymembers (Laskowski, M et al., 1980, Ann. Rev. Biochem. 49:593-626).Based on this known bonding pattern and the fact that the folding ofPlacental Bikunin (7-64) and (102-159) into active protease inhibitorsis accompanied by a mass reduction consistent with the formation ofthree intrachain disulfide bonds (Examples 2 and 1), it is highlyprobable that the disulfide bonding within the Kunitz domains ofPlacental Bikunin occur between cysteine residues: C11 and C61; C20 andC44; C36 and C57; C106 and C156; C115 and C139; C131 and C152.Furthermore, this pattern of disulfide bonding is highly probable inlarger forms of Placental Bikunin containing both Kunitz domains sincesuch forms of the protein are also active serine protease inhibitors andbecause N-terminal sequencing (Example 7) of native Placental Bikuninfor 50 cycles yielded a sequence that was silent at positions where thecysteine residues were expected.

The placental bikunin, isolated domains or other variants of the presentinvention may be produced by standard solid phase peptide synthesisusing either t-Boc chemistry as described by Merrifield R. B. and BaranyG., in: The peptides, Analysis, Synthesis, Biology, 2, Gross E. et al.,Eds. Academic Press (1980) Chapter 1; or using F-moc chemistry asdescribed by Carpino L. A., and Han G. Y., (1970) J. Amer Chem Soc,92,5748-5749, and illustrated in Example 2. Alternatively, expression ofa DNA encoding the placental bikunin variant may be used to producerecombinant placental bikunin variants.

The invention also relates to DNA constructs that encode the Placentalbikunin protein variants of the present invention. These constructs maybe prepared by synthetic methods such as those described in Beaucage S.L. and Caruthers M. H., (1981) Tetrahedron Lett, 22, pp1859-1862;Matteucci M. D and Caruthers M. H., (1981), J. Am. Chem. Soc. 103, p3185; or from genomic or cDNA which may have been obtained by screeninggenomic or cDNA libraries with cDNA probes designed to hybridize withplacental bikunin encoding DNA sequence. Genomic or cDNA sequence can bemodified at one or more sites to obtain cDNA encoding any of the aminoacid substitutions or deletions described in this disclosure.

The instant invention also relates to expression vectors containing theDNA constructs encoding the placental bikunin, isolated domains or othervariants of the present invention that can be used for the production ofrecombinant placental bikunin variants. The cDNA should be connected toa suitable promoter sequence which shows transcriptional activity in thehost cell of choice, possess a suitable terminator and apoly-adenylation signal. The cDNA encoding the placental bikunin variantcan be fused to a 51 signal peptide that will result in the proteinencoded by the cDNA to undergo secretion. The signal peptide can be onethat is recognized by the host organism. In the case of a mammalian hostcell, the signal peptide can also be the natural signal peptide presentin full length placental bikunin. The procedures used to prepare suchvectors for expression of placental bikunin variants are well known inthe art and are for example described in Sambrook et al., MolecularCloning: A laboratory Manual, Cold Spring Harbor, N.Y., (1989).

The instant invention also relates to transformed cells containing theDNA constructs encoding the placental bikunin, isolated domains or othervariants of the present invention that can be used for the production ofrecombinant placental bikunin variants. A variety of combinations ofexpression vector and host organism exist which can be used for theproduction of the placental bikunin variants. Suitable host cellsinclude baculovirus infected Sf9 insect cells, mammalian cells such asBHK, CHO, Hela and C-127, bacteria such as E. coli, and yeasts such asSaccharomyces cervisiae. Methods for the use of mammalian, insect andmicrobial expressions systems needed to achieve expression of placentalbikunin are well known in the art and are described, for example, inAusubel F. M et al., Current Protocols in Molecular Biology, John Wiley& Sons (1995), Chapter 16. For fragments of placental bikunin containinga single Kunitz inhibitor domain such as bikunin (7-64) and (102-159),yeast and E. coli expression systems are preferable, with yeast systemsbeing most preferred. Typically, yeast expression would be carried outas described in U.S. Pat. No. 5,164,482 for aprotinin variants andadapted in Example 5 of the present specification for placental bikunin(102-159). E. coli expression could be carried out using the methodsdescribed in U.S. Pat. No. 5,032,573. Use of mammalian and yeast systemsare most preferred for the expression of larger placental bikuninvariants containing both inhibitor domains such as the variant bikunin(7-159).

DNA encoding variants of placental bikunin that possess amino acidsubstitution of the natural amino sequence can be prepared forexpression of recombinant protein using the methods of Kunkel T. A.,(1985) Proc. Natl. Acad. Sci USA 82: 488-492. Briefly, the DNA to bemutagenized is cloned into a single stranded bacteriophage vector suchas M13. An oligonucleotide spanning the region to be changed andencoding the substitution is hybridized to the single stranded DNA andmade double stranded by standard molecular biology techniques. This DNAis then transformed into an appropriate bacterial host and verified bydideoxynucleotide sequencing. The correct DNA is then cloned into theexpression plasmid. Alternatively, the target DNA may be mutagenized bystandard PCR techniques, sequenced, and inserted into the appropriateexpression plasmid.

The following particular examples are offered by way of illustration,and not limitation, of certain aspects and preferred embodiments of theinstant invention.

EXAMPLE 1

Preparation of Synthetic Placental Bikunin (102-159)

Materials and methods/Reagents used. The fluorogenic substrateTos-Gly-Pro-Lys-AMC was purchased from Bachem BioScience Inc (King ofPrussia, Pa.). PNGB, Pro-Phe-Arg-AMC, Ala-Ala-Pro-Met-AMC, bovinetrypsin (type III), human plasma kallikrein, and human plasmin were fromSigma (St. Louis, Mo.).

Recombinant aprotinin (Trasylol®) was from Bayer AG (Wuppertal,Germany). Pre-loaded Gin Wang resin was from Novabiochem (La Jolla,Calif.). Thioanisole, ethanedithiol and t-butyl methyl ether was fromAldrich (Milwaukee, Wis.).

Quantification of Functional Placental Bikunin (7-64) and (102-159)

The amount of trypsin inhibitory activity present in the refolded sampleat various stages of purification was measured using GPK-AMC as asubstrate. Bovine trypsin (200 pmoles) was incubated for 5 min at 37% Cwith bikunin (7-64) or (102-159), from various stages of purification,in buffer A (50 mM Hepes, pH 7.5, 0.1 M NaCl, 2 mM CaCl₂ and 0.01%triton X-100). GPK-AMC was added (20 μM final) and the amount ofcoumarin produced was determined by measuring the fluorescence (ex=370nm, em=432 nm) on a Perkin-Elmer LS-50B fluorimeter over a 2 min.period. For samples being tested the % inhibition for each wascalculated according to equation 1; where R_(o) is the rate offluorescence increase in the presence of inhibitor and R₁ is the ratedetermined in the absence of added sample. One unit of activity for theinhibitor is defined as the amount needed to achieve 50% inhibition inthe assay using the conditions as described.% inhibition=100×[1−R _(o) /R ₁]  (1)

Synthesis. Placental bikunin (102-159) was synthesized on an AppliedBiosystems model 420A peptide synthesizer using NMP-HBTU Fmoc chemistry.The peptide was synthesized on pre loaded Gin resin with an 8-foldexcess of amino acid for each coupling. Cleavage and deprotection wasperformed in 84.6% trifluoroacetic acid (TFA), 4.4% thioanisole, 2.2%ethanedithiol, 4.4% liquified phenol, and 4.4% H₂O for 2 hours at roomtemperature. The crude peptide was precipitated, centrifuged and washedtwice in t-butyl methyl ether. The peptide was purified on a Dynamax 60AC 18 reverse-phase HPLC column using a TFA/acetonitrile gradient. Thefinal preparation (61.0 mg) yielded the correct amino acid compositionand molecular mass by Electrospray mass spectroscopy (MH+=6836.1;calcd=6835.5) for the predicted sequence: (SEQ ID NO: 6) YEEYCTANAVTGPCRASFPR WYFDVERNSC NNFIYGGCRG NKNSYRSEEA CMLRCFRQ

Purification. Refolding of placental bikunin (102-159) was performedaccording to the method of Tam et al., (J. Am. Chem. Soc. 1991,113:6657-62). A portion of the purified peptide (15.2 mg) was dissolved in4.0 ml of 0.1 M Tris, pH 6.0, and 8 M urea. Oxidation of the disulfideswas accomplished by dropwise addition of a solution containing 23% DMSO,and 0.1 M Tris, pH 6.0 to obtain a final concentration of 0.5 mg/mlpeptide in 20% DMSO, 0.1 M Tris, pH 6.0, and 1 M urea. The solution wasallowed to stir for 24 hr at 25° C. after which it was diluted 1:10 inbuffer containing 50 mM Tris, pH 8.0, and 0.1 M NaCl. The material waspurified using a kallikrein affinity column made by covalently attaching30 mg of bovine pancreatic kallikrein (Bayer AG) to 3.5 mls of CNBractivated Sepharose (Pharmacia) according to the manufacturersinstructions. The refolded material was loaded onto the affinity columnat a flow rate of 1 ml/min and washed with 50 mM Tris, pH 8.0, and 0.1 MNaCl until absorbance at 280 nm of the wash could no longer be detected.The column was eluted with 3 volumes each of 0.2 M acetic acid, pH 4.0and 1.7. Active fractions were pooled (see below) and the pH of thesolution adjusted to 2.5. The material was directly applied to a VydacC18 reverse-phase column (5 micron, 0.46×25 cm) which had beenequilibrated in 22.5% acetonitrile in 0.1% TFA. Separation was achievedusing a linear gradient of 22.5 to 40% acetonitrile in 0.1% TFA at 1.0ml/min over 40 min. Active fractions were pooled, lyophilized,redissolved in 0.1% TFA, and stored at −20° C. until needed.

Results. Synthetic placental bikunin (102-159) was refolded using 20%DMSO as the oxidizing agent as described above, and purified by a 2-steppurification protocol as shown below, to yield an active trypsininhibitor (Table 1 below). TABLE 1 Purification table for the isolationof synthetic placental bikunin (102-159) TABLE 1 Purification VolUnits^(c) SpA Step (ml) Mg/ml Mg (U) (U/mg) Yield 8.0 M Urea 4.03.75^(a) 15.0 0 0 — 20% DMSO 32.0 0.47^(a) 15.0 16,162 1,078 100Kallikrein 9.8 0.009^(b) 0.09 15,700 170,000 97 affinity C18 3.00.013^(ab) 0.04 11,964 300,000 74^(a)Protein determined by AAA.^(b)Protein determined by OD280 nm using the extinction coefficientdetermined for the purified protein (1.7 × 10⁴ Lmol⁻¹ cm⁻¹).^(c)One Unit is defined as the amount of material required to inhibit50% of trypsin activity in a standard assay.

Chromatography of the crude refolded material over an immobilized bovinepancreatic kallikrein column selectively isolated 6.0% of the proteinand 97% of the trypsin inhibitory activity present. Subsequentchromatography using C18 reverse-phase yielded a further purification of2-fold, with an overall recovery of 74%. On RPHPLC, the reduced andrefolded placental bikunin (102-159), exhibited elution times of 26.3and 20.1 minutes, respectively. Mass spectroscopy analysis of thepurified material revealed a molecular mass of 6829.8; a loss of 6 massunits from the starting material. This demonstrates the completeformation of the 3 disulfides predicted from the peptide sequence.

The isoelectric points of the purified, refolded synthetic placentalbikunin (102-159) was determined using a Multiphor II ElectrophoresisSystem (Pharmacia) run according to the manufacturers suggestions,together with pI standards, using a precast Ampholine® PAGplate (pH 3.5to 9.5) and focused for 1.5 hrs. After staining, the migration distancefrom the cathodic edge of the gel to the different protein bands wasmeasured. The pI of each unknown was determined by using a standardcurve generated by a plot of the migration distance of standards versusthe corresponding pI's. With this technique, the pI of placental bikunin(102-159) was determined to be 8.3, in agreement with the valuepredicted from the amino acid sequence. This is lower than the value of10.5 established for the pI of aprotinin. (Tenstad et al., 1994, ActaPhysiol. Scand. 152: 33-50).

EXAMPLE 2

Preparation of Synthetic Placental Bikunin (7-64)

Placental bikunin (7-64) was synthesized, refolded and purifiedessentially as described for placental bikunin (102-159) but with thefollowing modifications: during refolding, the synthetic peptide wasstirred for 30 hr as a solution in 20% DMSO at 25° C.; purification byC18 RP-HPLC was achieved with a linear gradient of 25 to 45%acetonitrile in 0.1% TFA over 40 min (1 ml/min). Active fractions fromthe first C18 run were reapplied to the column and fractionated with alinear gradient (60 min, 1 ml/min) of 20 to 40% acetonitrile in 0.1%TFA.

Results. The final purified reduced peptide exhibited an MH+=6563,consistent with the sequence: (SEQ ID NO: 4) IHDFCLVSKV VGRCRASMPRWWYNVTDGSC QLFVYGGCDG NSNNYLTKEE CLKKCATV

The refolding and purification yielded a functional Kunitz domain thatwas active as an inhibitor of trypsin (Table 2 below). TABLE 2APurification table for the isolation of synthetic placental bikunin(7-64) TABLE 2A Purification Vol Units^(c) SpA Step (ml) Mg/ml Mg (U)(U/mg) Yield 8.0 M Urea 8.0 2.5 20.0 0 0 — 20% DMSO 64.0 0.31 20.068,699 3,435 100 Kall affinity 11.7 0.10 1.16 43,333 36,110 62 pH 4.0Kall affinity 9.0 0.64 0.06 21,905 350,143 31.9 pH 1.7 C18-1 4.6 0.140.06 21,905 350,143 31.9 C18-2 1.0 0.08 0.02 7,937 466,882 11.5

The purified refolded protein exhibited an MH+=6558, i.e. 5±1 mass unitsless than for the reduced peptide. This demonstrates that refoldingcaused the formation of at least one appropriate disulfide bond.

The pI of placental bikunin (7-64) was determined using the methodsemployed to determine the pI of placental bikunin (102-159). Placentalbikunin (7-64) exhibited a pI that was much higher than the predictedvalue (pI=7.9). Refolded placental bikunin (7-64) migrated to thecathodic edge of the gel (pH 9.5) and an accurate pI could not bedetermined under these conditions.

Continued Preparation of Synthetic Placental Bikunin (7-64)

Because the synthetic placental bikunin (7-64) may not have undergonecomplete deprotection prior to purification and refolding, refolding wasrepeated using protein which was certain to be completely deprotected.Placental bikunin (7-64) was synthesized, refolded and purifiedessentially as described for placental bikunin (102-159) but with thefollowing modifications: during refolding, the synthetic peptide (0.27mg/ml) was stirred for 30 hr as a solution in 20% DMSO at 25 C;purification by C18 RP-HPLC was achieved with a linear gradient of 22.5to 50% acetonitrile in 0.1% TFA over 40 min (1 ml/min).

Results. The final purified reduced peptide exhibited an MH+=6567.5,consistent with the sequence: (SEQ ID NO: 4) IHDFCLVSKV VGRCRASMPRWWYNVTDGSC QLFVYGGCDG NSNNYLTKEE CLKKCATV

The refolding and purification yielded a functional Kunitz domain thatwas as active as an inhibitor of trypsin (Table 2B below). TABLE 2BPurification table for the isolation of synthetic placental bikunin(7-64) TABLE 2B Purification Vol Units^(c) SpA Step (ml) Mg/ml Mg (U)(U/mg) Yield 8.0 M Urea 4.9 2.1 10.5 0 0 — 20% DMSO 39.0 0.27 10.5236,000 22,500 100 Kallikrein 14.5 0.3 0.43 120,000 279,070 50.9affinity (pH 2) C18 Reverse- 0.2 1.2 0.24 70,676 294,483 30.0 Phase

The purified refolded protein exhibited an MH+=6561.2, i.e. 6.3 massunits less than for the reduced peptide. This demonstrates thatrefolding caused the formation of the expected three disulfide bonds.

The pI of refolded placental bikunin (7-64) was determined using themethods employed to determine the pI of placental bikunin (102-159).Refolded placental bikunin (7-64) exhibited a pI of 8.85, slightlyhigher than the predicted value (pI=7.9).

EXAMPLE 3

In Vitro Specificity of Functional Placental Bikunin Fragment (102-159)

Proteases. Bovine trypsin, human plasmin, and bovine pancreatickallikrein quantitation was carried out by active site titration usingp-nitrophenyl p′-guanidinobenzoate HCl as previously described (Chase,T., and Shaw, E., (1970) Methods Enzmol., 19: 20-27). Human kallikreinwas quantitated by active site titration using bovine aprotinin as astandard and PFR-AMC as a substrate assuming a 1:1 complex formation.The K_(m) for GPK-AMC with trypsin and plasmin under the conditions usedfor each enzyme was 29 μM and 726 μM, respectively; the K_(m) forPFR-AMC with human plasma kallikrein and bovine pancreatic kallikreinwas 457 μM and 81.5 μM, respectively; the K_(m) for AAPR-AMC withelastase was 1600 μM. Human tissue kallikrein (Bayer, Germany)quantification was carried out by active site titration usingp′nitrophenyl p′-guanidinobenzoate HCl as previously described (Chase,T., and Shaw, E., (1970) Methods Enzmol. 19: 20-27).

Inhibition Kinetics: The inhibition of trypsin by placental bikunin(102-159) or aprotinin was measured by the incubation of 50 pM trypsinwith placental bikunin (102-159) (0-2 nM) or aprotinin (0-3 nM) inbuffer A in a total volume of 1.0 ml. After 5 min. at 37° C., 15 μl of 2mM GPK-AMC was added and the change in fluorescence (as above) wasmonitored. The inhibition of human plasmin by placental bikunin(102-159) and aprotinin was determined with plasmin (50 pM) andplacental bikunin (102-159) (0-10 nM) or aprotinin (0-4 nM) in buffercontaining 50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, and 0.02% triton x-100.After 5 min. incubation at 37° C., 25 μl of 20 mM GPK-AMC was added andthe change in fluorescence monitored. The inhibition of human plasmakallikrein by placental bikunin (102-159) or aprotinin was determinedusing kallikrein (2.5 nM) and placental bikunin (102-159) (0-3 nM) oraprotinin (0-45 nM) in 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, and 0.02%triton x-100. After 5 min. at 37° C. 15 μl of 20 mM PFR-AMC was addedand the change in fluorescence monitored. The inhibition of bovinepancreatic kallikrein by placental bikunin (102-159) and aprotinin wasdetermined in a similar manner with kallikrein (92 pM), placentalbikunin (102-159) (0-1.6 nM) and aprotinin (0-14 pM) and a finalsubstrate concentration of 100 μM. The apparent inhibition constantK_(i)* was determined using the nonlinear regression data analysisprogram Enzfitter software (Biosoft, Cambridge, UK): The kinetic datafrom each experiment were analyzed in terms of the equation for a tightbinding inhibitor:V _(i) /V _(o)=1−(E _(o) +I _(o) +K _(i)*−[(E _(o) +I _(o) +K _(i)*)²−4E_(o) I _(o)]^(1/2))/2E _(o)  (2)where V_(i)/V_(o) is the fractional enzyme activity (inhibited vs.uninhibited rate), and E_(o) and I_(o) are the total concentrations ofenzyme and inhibitor, respectively. Ki values were obtained bycorrecting for the effect of substrate according to the equation:K _(i) =K _(i)*/(1+[S _(o) ]/K _(m))  (3)(Boudier, C, and Bieth, J. G., (1989) Biochim Biophys Acta., 995: 36-41)

For the inhibition of human neutrophil elastase by placental bikunin(102-159) and aprotinin, elastase (19 nM) was incubated with placentalbikunin (102-159) (150 nM) or aprotinin (0-7.5 μM) in buffer containing0.1 M Tris-HCl (pH 8.0), and 0.05% triton X-100. After 5 min at 37% C,AAPM-AMC (500 μM or 1000 μM) was added and the fluorescence measuredover a two-minute period. Ki values were determined from Dixon plots ofthe form 1/V versus [I] performed at two different substrateconcentrations (Dixon et al., 1979).

The inhibition of human tissue kallikrein by aprotinin, placentalbikunin fragment (7-64) or placental bikunin fragment (102-159) wasmeasured by the incubation of 0.35 nM human tissue kallikrein withplacental bikunin (7-64) (0-40 nM) or placental bikunin (102-159) (0-2.5nM), or aprotinin (0-0.5 nM) in a 1 ml reaction volume containing 50 mMTris-HCl buffer pH 9.0, 50 mM NaCl, and 0.1% triton x-100. After 5 min.at 37° C., 5 ul of 2 mM PFR-AMC was added achieving 10 uM final and thechange in fluorescence monitored. The Km for PFR-AMC with human tissuekallikrein under the conditions employed was 5.7 uM. The inhibition ofhuman factor Xa (American Diagnostica, Inc, Greenwich, Conn.) bysynthetic placental bikunin (102-159), recombinant placental bikunin,and aprotinin was measured by the incubation of 0.87 nM human factor Xawith increasing amounts of inhibitor in buffer containing 20 mM Tris (pH7.5), 0.1 M NaCl, and 0.1% BSA. After 5 min. at 37° C., 30 ul of 20 mMLGR-AMC (Sigma) was added and the change in fluorescence monitored. Theinhibition of human urokinase (Sigma) by Kunitz inhibitors was measuredby the incubation of urokinase (2.7 ng) with inhibitor in a total volumeof 1 ml buffer containing 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, and 0.1%Triton x-100. After 5 min. at 37° C., 35 ul of 20 mM GGR-AMC (Sigma) wasadded and the change in fluorescence monitored. The inhibition of FactorXIa (from Enzyme Research Labs, Southbend, Ind.) was measured byincubating FXIa (0.1 nM) with either 0 to 800 nM placental bikunin(7-64), 0 to 140 nM placental bikunin (102-159) or 0 to 40 uM aprotininin buffer containing 50 mM Hepes pH 7.5, 100 mM NaCl, 2 mM CaCl2, 0.01%triton x-100, and 1% BSA in a total volume of 1 ml. After 5 min at 37 C,10 ul of 40 mM Boc-Glu(OBzl)-Ala-Arg-AMC (Bachem Biosciences, King ofPrussia, Pa.) was added and the change in fluorescence monitored.

Results: A direct comparison of the inhibition profiles of placentalbikunin (102-159) and aprotinin was made by measuring their inhibitionconstants with various proteases under identical conditions. The K_(i)values are listed in Table 3 below. TABLE 3 Ki values for the inhibitionof various proteases by bikunin (102-159) TABLE 3 bikunin Protease(102-159) Aprotinin Substrate Km (concentration) Ki (nM) Ki (nM)(concentration) (mM) Trypsin (48.5 pM) 0.4 0.8 GPK-AMC 0.022 (0.03 mM)Chymotrypsin 0.24 0.86 AAPF-pMA 0.027 (5 nM) (0.08 mM) Bovine Pancreatic0.4 0.02 PFR-AMC 0.08 Kallikrein (0.1 mM) (92.0 pM) Human Plasma 0.319.0 PFR-AMC 0.46 Kallikrein (0.3 mM) (2.5 nM) Human Plasmin 1.8 1.3GPK-AMC 0.73 (50 pM) (0.5 mM) Human Neutrophil 323.0 8500.0 AAPM-AMC 1.6Elastase (19 nM) (1.0 μM) Factor XIIa >300.0 12,000.0 PFR-AMC 0.35 (0.2μM) Human Tissue 0.13 0.004 PFR-AMC 0.0057 Kallikrein (10 μM) (0.35 nM)factor Xa 274 N.I. at LGR-AMC N.D. (0.87 nM) 3 μM (0.6 mM) urokinase11000 4500 GGR-AMC H.D. (0.7 mM) factor XIa 15 288 E(OBz)AR- 0.46 (0.1nM) AMC (0.4 mM)

Placental bikunin (102-159) and aprotinin inhibit bovine trypsin andhuman plasmin to a comparable extent under the conditions employed.Aprotinin inhibited elastase with a Ki of 8.5 μM. Placental bikunin(102-159) inhibited elastase with a Ki of 323 nM. The K_(i) value forthe placental bikunin (102-159) inhibition of bovine pancreatickallikrein was 20-fold higher than that of aprotinin inhibition. Incontrast, placental bikunin (102-159) is a more potent inhibitor ofhuman plasma kallikrein than aprotinin and binds with a 56-fold higheraffinity.

Because placental bikunin (102-159) is greater than 50 times more potentthan Trasylol® as an inhibitor of kallikrein, smaller amounts of humanplacental bikunin, or fragments thereof (i.e. placental bikunin(102-159)) are needed than Trasylol® in order to maintain the effectivepatient doses of inhibitor in KIU. This reduces the cost per dose of thedrug and reduces the likelihood of adverse nephrotoxic effects uponre-exposure of the medicament to patients. Furthermore, the protein ishuman derived, and thus much less immunogenic in man than aprotininwhich is derived from cows. This results in significant reductions inthe risk of incurring adverse immunologic events upon re-exposure of themedicament to patients.

EXAMPLE 4

In Vitro Specificity of Functional Placental Bikunin Fragment (7-64)

In vitro specificity of functional human placental bikunin (7-64) wasdetermined using the materials and methods as described in the Examplesabove.

Results: The table below shows the efficacy of placental bikunin (7-64)as an inhibitor of various serine proteases in vitro. Data is showncompared against data obtained for screening inhibition using eitherplacental bikunin (102-159), or aprotinin (Trasylol®) TABLE 4A Ki valuesfor the inhibition of various proteases by bikunin (7-64) TABLE 4Abikunin Protease bikunin (7-64) Aprotinin (102-159) (concentration) Ki(nM) Ki (nM) Ki (nM) Trypsin (48.5 pM) 0.17 0.8 0.4 Bovine Pancreatic0.4 0.02 0.4 Kallikrein (92.0 pM) Human Plasma 2.4 19.0 0.3 Kallikrein(2.5 nM) Human Plasmin (50 pM) 3.1 1.3 1.8 Bovine chymotrypsin (5 nM)0.6 0.9 0.2 Factor XIIa >300 12,000 >300 elastase >100 8500 323

The results show that the amino acid sequence encoding placental bikunin(7-64) can be refolded to obtain an active serine protease inhibitorthat is effective against at least four trypsin-like serine proteases.

Table 4B below also shows the efficacy of refolded placental bikunin(7-64) as an inhibitor of various serine proteases in vitro. Refoldedplacental bikunin (7-64) was prepared from protein that was certain tobe completely deprotected prior to purification and refolding. Data isshown compared against data obtained for screening inhibition usingeither placental bikunin (102-159), or aprotinin (Trasylol®). TABLE 4BKi values for the inhibition of various proteases by bikunin (7-64)TABLE 4B bikunin Protease bikunin (7-64) Aprotinin (102-159)(concentration) Ki (nM) Ki (nM) Ki (nM) Trypsin (50 pM) 0.2 0.8 0.3Human Plasma 0.7 19.0 0.7 Kallikrein (0.2 nM) Human Plasmin (50 pM) 3.71.3 1.8 Factor XIIa Not done 12,000 4,500 Factor XIa (0.1 nM) 200 288 15Human Tissue Kallikrein 2.3 0.004 0.13

Suprisingly, placental bikunin (7-64) was more potent than aprotinin atinhibiting human plasma kallikrein, and at least similar in efficacy asa plasmin inhibitor. These data show that placental bikunin (7-64) is atleast as effective as aprotinin, using in vitro assays, and that onewould expect better or similar potency in vivo.

EXAMPLE 5

Expression of Placental Bikunin Variant (102-159) in Yeast

The DNA sequence encoding placental bikunin 102-159 (SEQ ID NO: 6) wasgenerated using synthetic oligonucleotides. The final DNA productconsisted (5′ to 3′) of 15 nucleotides from the yeast a-mating factorpropeptide sequence fused to the in-frame cDNA sequence encodingplacental bikunin (102-159), followed by an in-frame stop codon. Uponcloning into a yeast expression vector pS604, the cDNA would direct theexpression of a fusion protein comprising an N-terminal yeast a-matingfactor propeptide fused to the 58 amino acid sequence of placentalbikunin (102-159). Processing of this fusion protein at a KEX-2 cleavagesite at the junction between the a-mating factor and Kunitz domain wasdesigned to liberate the Kunitz domain at its native N-terminus.

A 5′ sense oligonucleotide of the following sequence and containing aHindIII site for cloning was synthesized: (SEQ ID NO: 42) GAA GGG GTAAGC TTG GAT AAA AGA TAT GAA GAA TAC TGC ACC GCC AAC GCA GTC ACT GGG CCTTGC CGT GCA TCC TTC CCA CGC TGG TAC TTT GAC GTG GAG AGG

A 3′ antisense oligonucleotide of the following sequence and containingboth a BamHI site for cloning and a stop codon was synthesized: (SEQ IDNO: 43) CGC GGA TCC CTA CTG GCG GAA GCA GCG GAG CAT GCA GGC CTC CTC AGAGCG GTA GCT GTT CTT ATT GCC CCG GCA GCC TCC ATA GAT GAA GTT ATT GCA GGAGTT CCT CTC CAC GTC AAA GTA CCA GCG

The oligonucleotides were dissolved in 10 mM Tris buffer pH 8.0containing 1 mM EDTA, and 12 ug of each oligo were added combined andbrought to 0.25M NaCl. To hybridize, the oligonucleotides were denaturedby boiling for 5 minutes and allowed to cool from 65° C. to room tempover 2 hrs. Overlaps were extended using the Klenow fragment anddigested with Hindin and BamHI. The resulting digested double strandedfragment was cloned into pUC19 and sequence confirmed. A clonecontaining the fragment of the correct sequence was digested withBamHI/HindIII to liberate the bikunin containing fragment with thefollowing+strand sequence: (SEQ ID.: 44) GAA GGG GTA AGC TTG GAT AAA AGATAT GAA GAA TAC TGC ACC GCC AAC GCA GTC ACT GGG CCT TGC CGT GCA TCC TTCCCA CGC TGG TAC TTT GAC GTG GAG AGG AAC TCC TGC AAT AAC TTC ATC TAT GGAGGC TGC CGG GGC AAT AAG AAC AGC TAC CGC TCT GAG GAG GCC TGC ATG CTC CGCTGC TTC CGC CAG TAG GGA TCCwhich was then gel purified and ligated into BamHI/HindIII cut pS604.The ligation mixture was extracted into phenol/chloroform and purifiedover a S-200 minispin column. The ligation product was directedtransformed into yeast strains SC101 and WHL341 and plated on uraselection plates. Twelve colonies from each strain were re-streaked onura drop out plates. A single colony was inoculated into 2 ml of ura DOmedia and grown over night at 30° C. Cells were pelleted for 2 minutesat 14000×g and the supernatants evaluated for their content of placentalbikunin (102-159).Detection of Expression of Placental Bikunin (102-159) in TransformedYeast

Firstly, the supernatants (50 ul per assay) were evaluated for theircapacity to inhibit the in vitro activity of trypsin using the assaymethods as described in Example 1 (1 ml assay volume). An un-used mediaonly sample as well as a yeast clone expressing an inactive variant ofaprotinin served as negative controls. A yeast clone expressing naturalaprotinin served as a positive control and is shown for comparison.

The second method to quantify placental bikunin (102-159) expressionexploited use of polyclonal antibodies (pAbs) against the syntheticpeptide to monitor the accumulation of the recombinant peptide usingWestern blots. These studies were performed only with recombinantsderived from strain SC101, since these produced greater inhibitoryactivity than recombinants derived from strain WHL341.

To produce the pAb, two 6-8 week old New Zealand White female rabbits(Hazelton Research Labs, Denver, Pa.) were immunized on day zero with250 ug of purified reduced synthetic placental bikunin (102-159), inComplete Freund's adjuvant, followed by boosts on days 14, 35 and 56 and77 each with 125 ug of the same antigen in Incomplete Freund's adjuvant.Antiserum used in the present studies was collected after the thirdboost by established procedures. Polyclonal antibodies were purifiedfrom the antiserum over protein A.

Colonies 2.4 and 2.5 from transformation of yeast SC101 (FIG. 8) as wellas an aprotinin control were grown overnight in 50 ml of ura DO media at30° C. Cells were pelleted and the supernatant concentrated 100-foldusing a Centriprep 3 (Amicon, Beverly, Mass.) concentrator. Samples ofeach (30 μl) were subjected to SDS-PAGE on 10-20% tricine buffered gels(Novex, San Diego, Calif.) using the manufacturers procedures. Duplicategels were either developed with a silver stain kit (IntegratedSeparation Systems, Nantick, Mass.) or transferred to nitrocellulose anddeveloped with the purified polyclonal antibody elicited to syntheticbikunin (102-159). Alkaline-phosphatase conjugated goat anti-rabbitantibody was used as the secondary antibody according to themanufacturer's directions (Kirkegaard and Perry, Gaithersburg, Md.).

Purification of Placental Bikunin (102-159) from a Transformed Strain ofSC101

Fermentation broth from a 1 L culture of SC101 strain 2.4 was harvestedby centrifugation (4,000 g×30 min.) then applied to a 1.0 ml column ofanhydrochymotrypsin-sepharose (Takara Biochemical Inc., CA), that waspreviously equilibrated with 50 mM Hepes buffer pH 7.5 containing 0.1MNaCl, 2 mM CaCl₂ and 0.01% (v/v) triton X-100. The column was washedwith the same buffer but containing 1.0 M NaCl until the A280 nmdeclined to zero, whereupon the column was eluted with 0.1M formic acidpH 2.5. Eluted fractions were pooled and applied to a C18 column (Vydac,5 um, 4.6×250 mm) previously equilibrated with 0.1% TFA, and eluted witha 50 min. linear gradient of 20 to 80% acetonitrile in 0.1% TFA.Fractions containing placental bikunin (102-159) were pooled andre-chromatographed on C18 employing elution with a linear 22.5 to 50%acetonitrile gradient in 0.1% TFA.

Results. FIG. 8 shows the percent trypsin activity inhibited by twelvecolonies derived from the transformation of each of strains SC101 andWHL341. The results show that all twelve colonies of yeast strain SC101transformed with the trypsin inhibitor placental bikunin (102-159) hadthe ability to produce a substantial amount of trypsin inhibitoryactivity compared to the negative controls both of which showed noability to inhibit trypsin. The activity is therefore related to theexpression of a specific inhibitor in the placental bikunin variant(102-159) transformed cells. The yeast WHL341 samples contained minimaltrypsin inhibitory activity. This may be correlated to the slow growthobserved with this strain under the conditions employed.

FIG. 9 shows the SDS-PAGE and western analysis of the yeast SC101supernatants. Silver stained SDS-PAGE of supernatants derived fromrecombinant yeasts 2.4 and 2.5 expressing placental bikunin (102-159) aswell as from the yeast expressing aprotinin yielded a protein bandrunning at approximated 6 kDa, corresponding to the size expected foreach recombinant Kunitz inhibitor domain. Western analysis showed thatthe 6 kDa bands expressed by stains 2.4 and 2.5 reacted with the pAbelicited to placental bikunin (102-159). The same 6 kDa band in theaprotinin control did not react with the same antibody, demonstratingthe specificity of the antibody for the placental bikunin variant(102-159).

The final preparation of placental bikunin C-terminal domain was highlypure by silver-stained SDS-PAGE (FIG. 10). The overall recovery ofbroth-derived trypsin inhibitory activity in the final preparation was31%. N-terminal sequencing of the purified inhibitor indicated that 40%of the protein is correctly processed to yield the correct N-terminusfor placental bikunin (102-159) while about 60% of the materialcontained a portion of the yeast a-mating factor. The purified materialcomprised an active serine protease inhibitor exhibiting an apparent Kiof 0.35 nM for the in vitro inhibition of plasma kallikrein.

In conclusion, the accumulation both of a protease inhibitor activityand a protein immunochemically related to synthetic bikunin (102-159) infermentation broth as well as the isolation of placental bikunin(102-159) from one of the transformed lines provided proof of expressionof placental bikunin in the recombinant yeast strains described herein,showing for the first time the utility of yeasts for the production ofplacental bikunin fragments.

Additional constructs were prepared in an effort to augment theexpression level of the Kunitz domain contained within placental bikunin102-159, as well as to increase the yield of protein with the correctN-terminus. We hypothesized that the N-terminal residues of placentalbikunin 102-159 (YEEY-) may have presented a cleavage site that is onlypoorly recognized by the yeast KEX-2 protease that enzymically removesthe yeast a-factor pro-region. Therefore, we prepared yeast expressionconstructs for the production of placental bikunin 103-159 (N-terminusof EEY . . . ), 101-159 (N-terminus of NYEEY . . . ) and 98-159(DMFNYEEY . . . ) in order to modify the P′ subsites surrounding theKEX-2 cleavage site. To attempt to augment the levels of recombinantprotein expression, we also used the yeast preferred codons rather thanmammalian preferred codons in preparing some of the constructs describedbelow. The constructs were essentially prepared as described above forplacental bikunin 102-159 (defined as construct #1) but with thefollowing modifications:

Construct #2 placental bikunin 103-159, yeast codon usage A 5′ senseoligonucleotide (SEQ ID NO: 55) GAAGGGGTAA GCTTGGATAA AAGAGAAGAATACTGTACTG CTAATGCTGT TACTGGTCCA TGTAGAGCTT CTTTTCCAAG ATGGTACTTTGATGTTGAAA GA

and 3′ antisense oligonucleotide (SEQ ID NO: 56) ACTGGATCCT CATTGGCGAAAACATCTCAA CATACAGGCT TCTTCAGATC TGTAAGAATT TTTATTACCT CTACAACCACCGTAAATAAA ATTATTACAA GAATTTCTTT CAACATCAAA GTACCATCTwere manipulated as described for the production of an expressionconstruct (construct #1 above) for the expression of placental bikunin102-159

Construct #3 placental bikunin 101-159, yeast codon usage A 5′ senseoligonucleotide (SEQ ID NO: 57) GAAGGGGTAA GCTTGGATAA AAGAAATTACGAAGAATACT GTACTGCTAA TGCTGTTACT GGTCCATGTA GAGCTTCTTT TCCAAGATGGTACTTTGATG TTGAAAGAand the same 3′ antisense oligonucleotide as used for construct #2, weremanipulated as described for the production of an expression construct(construct #1 above) for the expression of placental bikunin 102-159.

Construct #4 placental bikunin 98-159, yeast codon usage A 5′ senseoligonucleotide (SEQ ID NO: 58) GAAGGGGTAA GCTTGGATAA AAGAGATATGTTTAATTACG AAGAATACTG TACTGCTAAT GCTGTTACTG GTCCATGTAG AGCTTCTTTTCCAAGATGGT ACTTTGATGT TGAAAGAand the same 3′ antisense oligonucleotide as used for construct #2, weremanipulated as described for the production of an expression construct(construct #1 above).

Yeast strain SC101 (MATα, ura 3-52, suc 2) was transformed with theplasmids containing each of the above cDNAs, and proteins were expressedusing the methods that were described above for the production ofplacental bikunin 102-159 with human codon usage. Approximately 250 mlof each yeast culture was harvested, and the supernatant fromcentrifugation (15 min×3000 RPM) separately subjected to purificationover 1 ml columns of kallikrein-sepharose as described above. Therelative amount of trypsin inhibitory activity in the applysate, theamount of purified protein recovered and the N-terminal sequence of thepurified protein were determined and are listed below in Table 7. TABLE7 Relative production levels of different proteins containing theC-terminal Kunitz domain of placental bikunin TABLE 7 Relative conc. ofinhibitor N-terminal sequencing: in applysate amount (pmol) sequenceComments Construct #2 103-159 None detected none none no expression #3101-159 25% inhibition none none low expression #4  98-159 93%inhibition 910 DMFNYE- good Expression correct product #1 102-159 82%inhibition 480 AKEEGV- expression of active incorrectly processedprotein

The results show that placental bikunin fragments of different lengthsthat contain the C-terminal Kunitz domain show wide variation incapacity to express functional secreted protein. Constructs expressingfragments 101-159 and 103-159 yielded little or low enzymic activity inthe supernatants prior to purification, and N-terminal sequencing of0.05 ml aliquots of each purified fraction yielded undetectable amountsof inhibitor. On the other hand expression either of placental bikunin102-159 or 98-159 yielded significant amounts of protease activity priorto purification. N-terminal sequencing however showed that the purifiedprotein recovered from expression of 102-159 was once again largelyincorrectly processed, exhibiting an N-terminus consistent withprocessing of the majority of the pre-protein at a site within the yeastα-mating factor pro-sequence. The purified protein recovered fromexpression of placental bikunin 98-159 however was processed entirely atthe correct site to yield the correct N-terminus. Furthermore, nearlytwice as much protein was recovered as compared to the recovery ofplacental bikunin 102-159. Placental bikunin 98-159 thus represents apreferred fragment length for the production of the C-terminal Kunitzdomain of placental bikunin by the α-mating factor pre-prosequence/KEX-2 processing system of S. cerevisiae.

EXAMPLE 6

Alternative Procedure for Yeast Expression

The 58 amino acid peptide derived from the R74593 translation productcan also be PCR amplified from either the R87894-R74593 PCR productcloned into the TA vector™ (Invitrogen, San Diego, Calif.) after DNAsequencing or from human placental cDNA. The amplified DNA product willconsist of 19 nucleotides from the yeast α-mating factor leader sequencemated to the R74593 sequence which codes for the YEEY-CFRQ (58 residues)so as to make the translation product in frame, constructing an α-matingfactor/Kunitz domain fusion protein. The protein sequence also containsa kex 2 cleavage which will liberate the Kunitz domain at its nativeN-terminus.

The 51 sense oligonucleotide which contains a HindIII site for cloningwill contain the following sequence: (SEQ ID NO: 30) GCCAAGCTTGGATAAAAGAT ATGAAGAAT ACTGCACCGC CAACGCA

The 3′ antisense oligonucleotide contains a BamHI site for cloning aswell as a stop codon and is of the following sequence: (SEQ ID NO: 31)GGGGATCCTC ACTGCTGGCG GAAGCAGCGG AGCAT

The full 206 nucleotide cDNA sequence to be cloned into the yeastexpression vector is of the following sequence: (SEQ ID NO: 32)CCAAGCTTGG ATAAAAGATA TGAAGAATAC TGCACCGCCA ACGCAGTCAC TGGGCCTTGCCGTGCATCCT TCCCACGCTG GTACTTTGAC GTGGAGAGGA ACTCCTGCAA TAACTTCATCTATGGAGGCT GCCGGGGCAA TAAGAACAGC TACCGCTCTG AGGAGGCCTG CATGCTGCGCTGCTTCCGCC AGCAGTGAGG ATCCCC

After PCR amplification, this DNA will be digested with HindIII, BamHIand cloned into the yeast expression vector pMT15 (see U.S. Pat. No.5,164,482, incorporated by reference in the entirety) also digested withHindIII and BamHI. The resulting plasmid vector is used to transformyeast strain SC 106 using the methods described in U.S. Pat. No.5,164,482. The URA 3+yeast transformants are isolated and cultivatedunder inducing conditions. The yield of recombinant Placental bikuninvariants is determined according to the amount of trypsin inhibitoryactivity that accumulated in the culture supernatants over time usingthe in vitro assay method described above. Fermentation broths arecentrifuged at 9000 rpm for 30 minutes. The supernatant is then filteredthrough a 0.4 then a 0.2 [μm filter, diluted to a conductivity of 7.5ms, and adjusted to pH 3 with citric acid. The sample is then batchabsorbed onto 200 ml of S-sepharose fast flow (Pharmacia) in 50 mMsodium citrate pH 3 and stirred for 60 min. The gel is subsequentlywashed sequentially with 2 L of each of: 50 mM sodium citrate pH 3.0; 50mM Tris-HCL pH 9.0; 20 mM HEPES pH 6.0. The washed gel is transferredinto a suitable column and eluted with a linear gradient of 0 to 1 Msodium chloride in 20 mM HEPES pH 6.0. Eluted fractions containing invitro trypsin inhibitory activity are then pooled and further purifiedeither by a) chromatography over a column of immobilized anhydrotrypsin(essentially as described in Example 2); b) by chromatography over acolumn of immobilized bovine kallikrein; or c) a combination ofconventional chromatographic steps including gel filtration and/oranion-exchange chromatography.

EXAMPLE 7

Isolation and Characterization of Native Human Placental Bikunin fromPlacenta

Bikunin protein was purified to apparent homogeniety from whole frozenplacenta (Analytical Biological Services, Inc, Wilmington, Del.). Theplacenta (740 gm) was thawed to room temperature and cut into 0.5 to 1.0cm pieces, placed on ice and washed with 600 ml PBS buffer. The wash wasdecanted and 240 ml of placenta pieces placed into a Waring blender.After adding 300 ml of buffer consisting of 0.1 M Tris (pH 8.0), and 0.1M NaCl, the mixture was blended on high speed for 2 min, decanted into750.0 ml centrifuge tubes, and placed on ice. This procedure wasrepeated until all material was processed. The combined slurry wascentrifuged at 4500×g for 60 minutes at 4° C. The supernatant wasfiltered through cheese cloth and the placental bikunin purified using akallikrein affinity column made by covalently attaching 70 mg of bovinepancreatic kallikrein (Bayer AG) to 5.0 mls of CNBr activated Sepharose(Pharmacia) according to manufacturers instruction. The material wasloaded onto the affinity column at a flow rate of 2.0 ml/min and washedwith 0.1 M Tris (pH 8.0), 0.1 M NaCl until absorbance at 280 nm of thewash could no longer be detected. The column was further washed with 0.1M Tris (pH 8.0), 0.5 M NaCl and then eluted with 3 volumes of 0.2 Macetic acid, pH 4.0. Fractions containing kallikrein and trypsininhibitory (see below) activity were pooled, frozen, and lyophilized.Placental bikunin was further purified by gel-filtration chromatographyusing a Superdex 75 10/30 (Pharmacia) column attached to a BeckmanSystem Gold HPLC system. Briefly, the column was equilibrated in 0.1 MTris, 0.15 M NaCl, and 0.1% Triton X-100 at a flow rate of 0.5 ml/min.The lyophilized sample was reconstituted in 1.0 ml of 0.1 M Tris, pH 8.0and injected onto the gel-filtration column in 200 μl aliquots.Fractions were collected (0.5 ml) and assayed for trypsin and kallikreininhibitory activity. Active fractions were pooled, and the pH of thesolution adjusted to 2.5 by addition of TFA. The material was directlyapplied to a Vydac C18 reverse-phase column (5 micron, 0.46×25 cm) whichhad been equilibrated in 20% acetonitrile in 0.1% TFA. Separation wasachieved using a linear gradient of 20 to 80% acetonitrile in 0.1% TFAat 1.0 ml/min over 50 minutes after an initial 20 minute wash at 20%acetonitrile in 0.1% TFA. Fractions (1 ml) were collected and assayedfor trypsin and kallikrein inhibitory activity. Fractions containinginhibitory activity were concentrated using a speed-vac concentrator(Savant) and subjected to N-terminal sequence analysis.

Functional Assays for Placental Bikunin:

Identification of functional placental bikunin was achieved by measuringits ability to inhibit bovine trypsin and human plasma kallikrein.Trypsin inhibitory activity was performed in assay buffer (50 mM Hepes,pH 7.5, 0.1 M NaCl, 2.0 mM CaCl2, 0.1% Triton x-100) at room temperaturein a 96-well microtiter plate (Perkin Elmer) usingGly-Pro-Lys-Aminomethylcoumarin as a substrate. The amount of coumarinproduced by trypsin was determined by measuring the fluorescence (ex=370nm, em=432 nm) on a Perkin-Elmer LS-50B fluorimeter equipped with aplate reader. Trypsin (23 μg in 100 μl buffer) was mixed with 20 μl ofthe sample to be tested and incubated for 10 minutes at 25° C. Thereaction was started by the addition of 50 μl of the substrate GPK-AMC(33 μM final) in assay buffer. The fluorescence intensity was measuredand the % inhibition for each fraction was determined by:% inhibition=100×[1−Fo/F1]where Fo is the fluorescence of the unknown and F1 is the fluorescenceof the trypsin only control. Kallikrein inhibitory activity of thefractions was similarly measured using 7.0 nM kallikrein in assay buffer(50 mM Tris, pH 8.0, 50 mM NaCl, 0.1% triton x-100) and 66.0 μMPro-Phe-Arg-AMC as a substrate.Determination of the In Vitro Specificity of Placental Bikunin

The In vitro specificity of native human placental bikunin wasdetermined using the materials and methods as described in the precedingexamples above. Placental bikunin was quantified by active sitetitration against a known concentration of trypsin using GPK-AMC as asubstrate to monitor the fraction of unbound trypsin.

Protein Sequencing

The 1 ml fraction (C18-29 Delaria) was reduced to 300 ml in volume, on aSpeed Vac, to reduce the amount of organic solvent. The sample was thenloaded onto a Hewlett-Packard miniature biphasic reaction column, andwashed with 1 ml of 2% trifluoroacetic acid. The sample was sequenced ona Hewlett-Packard Model G1005A protein sequencing system using Edmandegradation. Version 3.0 sequencing methods and all reagents weresupplied by Hewlett-Packard. Sequence was confirmed for 50 cycles.

Results. Placental Bikunin was purified to apparent homogeniety bysequential kallikrein affinity, gel-filtration, and reverse-phasechromatography (see purification table below): TABLE 5 Purificationtable for native Placental Bikunin (1-179) TABLE 5 Vol OD 280 Units^(a)Units/OD Step (ml) (/ml) OD 280 (U) 280 Placenta 1800.0 41.7 75,0503,000,000 40.0 Supernatant Kallikrein 20.0 0.17 3.36 16,000 4,880Affinity pH 4.0 Kallikrein 10.2 0.45 4.56 12,000 2,630 Affinity pH 1.7Superdex 75 15.0 0.0085 0.13 3,191 24,546^(a)One Unit is defined as that amount which inhibits 50% of trypsinactivity in a standard assay.

The majority of the kallikrein and trypsin inhibitory activity elutedfrom the kallikrein affinity column in the pH 4.0 elution. Subsequentgel-filtration chromatography (FIG. 5) yielded a peak of kallikrein andtrypsin inhibitory activity with a molecular weight range of 10 to 40kDa as judged by a standard curve generated by running molecular weightstandards under identical conditions. Reverse-phase C18 chromatography(FIG. 6) yielded 4 peaks of inhibitory activity with the most potenteluting at approximately 30% acetonitrile. The activity associated withthe first peak to elute from C18 (fraction 29) exhibited an amino acidsequence starting with amino acid 1 of the predicted amino acid sequenceof placental bikunin (ADRER . . . ; SEQ ID NO: 1), and was identical tothe predicted sequence for 50 cycles of sequencing (underlined aminoacids in FIG. 3). Cysteine residues within this sequence stretch weresilent as expected for sequencing of oxidized protein. The cysteineresidues at amino acid positions 11 and 20 of mature placental bikuninwere later identified from sequencing of the S-pyridylethylated proteinwhereupon PTH-pyridylethyl-cysteine was recovered at cycles 11 and 20.

Interestingly, the asparagine at amino acid residue number 30 of thesequence (FIG. 3) was silent showing that this site is likely to beglycosylated. Fraction 29 yielded one major sequence corresponding tothat of placental bikunin starting at residue #1 (27 pmol at cycle 1)plus a minor sequence (2 pmol) also derived from placental bikuninstarting at residue 6 (SIHD . . . ). This shows that the finalpreparation sequenced in fraction 29 is highly pure, and most likelyresponsible for the protease inhibitory activity associated with thisfraction (FIG. 6).

Accordingly, the final preparation of placental bikunin from C18chromatography was highly pure based on a silver-stained SDS-PAGEanalysis (FIG. 7), where the protein migrated with an apparent Mr of 24kDa on a 10 to 20% acrylamide tricine gel (Novex, San Diego, Calif.)calibrated with the following molecular weight markers: insulin (2.9kDa); bovine trypsin inhibitor (5.8 kDa); lysozyme (14.7 kDa);β-lactaglobulin (18.4 kDa); carbonic anhydrase (29 kDa); and ovalbumin(43 kDa). The above size of placental bikunin on SDS-PAGE is consistentwith that predicted from the full length coding sequence (FIG. 4F).

As expected based on the N-terminal sequencing results described above,the purified protein reacted with an antibody elicited to placentalbikunin (7-64) to yield a band with the same Mr (FIG. 12A) as observedfor the purified preparation detected on gels by silver stain (FIG. 7).However, when the same preparation was reacted with an antibody elicitedto synthetic placental bikunin (102-159), a band corresponding to thefull length protein was not observed. Rather, a fragment thatco-migrated with synthetic bikunin (102-159) of approximately 6 kDa wasobserved. The simplest interpretation of these results is that thepurified preparation had undergone degradation subsequent topurification to yield an N-terminal fragment comprising the N-terminaldomain and a C-terminal fragment comprising the C-terminal domain.Assuming that the fragment reactive against antiserum to placentalbikunin (7-64) is devoid of the C-terminal end of the full lengthprotein, the size (24 kDa) would suggest a high state of glycosylation.

Table 6. below shows the potency of in vitro inhibition of variousserine proteases by placental bikunin. Data are compared with thatobtained with aprotinin (Trasylol®). TABLE 6 Ki values for theinhibition of various proteases by placental bikunin TABLE 6 ProteasePlacental Bikunin Aprotinin (concentration) Ki (nM) Ki (nM) Trypsin(48.5 pM) 0.13 0.8 Human Plasmin (50 pM) 1.9 1.3

The results show that placental bikunin isolated from a natural source(human placenta) is a potent inhibitor of trypsin-like serine proteases.

EXAMPLE 8

Expression Pattern of Placental Bikunin Amongst Different Human Organsand Tissues

A multiple tissue northern was purchased from Clontech which contained 2fig of polyA+ RNA from human heart, brain, placenta, lung; liver,skeletal muscle, kidney, and pancreas. Two different cDNA probes wereused: 1) a gel purified cDNA encoding placental bikunin (102-159); 2)the 780 base pair PCR-derived cDNA (FIG. 4E) liberated from a TA cloneby digestion with EcoRI and gel purified. Each probe was labeled using³²p-dCTp and a random priming labeling kit from Boehringer MannheimBiochemicals (Indiana), then used to hybridize to the multiple tissuenorthern according to the manufacturers specifications. Autoradiographswere generated using Biomax film with an 18 hr exposure time, anddeveloped using a Umax Scanner and scanned using Adobe Photoshop.

Results. The pattern of tissue expression observed using a placentalbikunin (102-159) probe (FIG. 11A) or a larger probe containing bothKunitz domains of placental bikunin (FIG. 11B) was essentially the sameas might be expected. The placental bikunin mRNA was most abundant inpancreas and placenta. Significant levels were also observed in lung,brain and kidney, while lower levels were observed in heart and liver,and the mRNA was undetectable in skeletal muscle. The transcript sizewas 1.95 kilobases in all cases, in close agreement with the predictedsize of placental bikunin deduced both from EST overlay and cloning offull length cDNA described in preceding sections.

The broad tissue distribution of the mRNA shows that placental bikuninis broadly expressed. Since the protein also contains a leader sequenceit would have ample exposure to the human immune system, requiring thatit become recognized as a self protein. Additional evidence for a broadtissue distribution of placental bikunin mRNA expression was derivedfrom the fact that some of the EST entries with homology to placentalbikunin (FIG. 4B) were derived from human adult and infant brain, andhuman retina, breast, ovary, olfactory epithelium, and placenta. It isconcluded therefore that administration of the native human protein tohuman patients would be unlikely to elicit an immune response.

Interestingly, the expression pattern of placental bikunin is somewhatreminiscent of that for bovine aprotinin which is found in high levelsin bovine lung and pancreas. To further elucidate the expression patternof placental bikunin, RT-PCR of total RNA from the following human cellswas determined: un-stimulated human umbilical vein endothelial cells(HUVECs), HK-2 (line derived from kidney proximal tubule), TF-1(erythroleukemia line) and phorbolester (PMA)-stimulated humanperipheral blood leukocytes. The probes used: CACCTGATCGCGAGACCCC;(sense; SEQ ID NO: 59) CTGGCGGAAGCAGCGGAGCATGC, (antisense; SEQ ID NO:60)were designed to amplify a 600 b.p placental bikunin encoding cDNAfragment. Comparisons were normalized by inclusion of actin primers toamplify an 800 b.p. actin fragment. Whereas the 800 b.p fragmentidentified on agarose gels with ethidium bromide was of equal intensityin all lanes, the 600 b.p. placental bikunin fragment was absent fromthe HUVECs but present in significant amounts in each of the other celllines. We conclude that placental bikunin is not expressed in at leastsome endothelial cells but is expressed in some leukocyte populations.

EXAMPLE 9

Purification and Properties of Placental Bikunin (1-170) Highly Purifiedfrom a Baculovirus/Sf9 Expression System

A large fragment of Placental bikunin containing both Kunitz domains(Placental Bikunin 1-170) was expressed in Sf9 cells as follows.Placental bikunin cDNA obtained by PCR (FIG. 4E) and contained within aTA vector (see previous Examples) was liberated by digestion withHindIII and XbaI yielding a fragment flanked by a 5′ XbaI site and 3′HindIII site. This fragment was gel purified and then cloned into theM13 mp19 vector (New England Biolabs, Beverly, Mass.). In vitromutagenesis (Kunkel T. A., (1985) Proc. Natl. Acad. Sci. USA, 82:488-492) was used to generate a Pst1 site 3′ to the XbaI site at the 5′end, but 5′ to the sequence encoding the ATG start site, naturalplacental bikunin signal peptide and mature placental bikunin codingsequence. The oligonucleotide used for the mutagenesis had the sequence:(SEQ ID NO: 61) 5′ CGC GTC TCG GCT GAC CTG GCC CTG CAG ATG GCG CAC GTGTGC GGG 3′

A stop codon (TAG) and Bg1II/XmaI site was similarly engineered at the3′ end of the cDNA using the oligonucleotide: (SEQ ID NO: 62) 5′ CTG CCCCTT GGC TCA AAG TAG GAA GAT CTT CCC CCC GGG GGG GTG GTT CTG GCG GGG CTG3′.

The stop codon was in frame with the sequence encoding placental bikuninand caused termination immediately following the Lysine at amino acidresidue 170, thus encoding a truncated placental bikunin fragment devoidof the putative transmembrane domain. The product from digestion withPst1 and BglII was isolated and cloned into the BacPac8 vector forexpression of Placental bikunin fragment (1-170) which contains bothKunitz domains but which is truncated immediately N-terminal to theputative transmembrane segment.

The expression of Bikunin by Sf-9 insect cells was optimal at amultiplicity of infection of 1 to 1 when the medium was harvested at 72h post infection. After harvesting, the baculovirus cell culturesupernatant (2 L) was adjusted to pH 8.0 by the addition of Tris-HCl.Bikunin was purified by chromatography using a 5 ml bovine pancreatickallikrein affinity column as previously described in Example 7 for thepurification of native placental bikunin from placenta. Eluted materialwas adjusted to pH 2.5 with TFA and subjected to chromatography on a C18reverse-phase column (1.0×25 cm) equilibrated in 10% acetonitrile in0.1% TFA at a flow rate of 1 ml/min. The bikunin was eluted with alinear gradient of 10 to 80% acetonitrile in 0.1% TFA over 40 min.Active fractions were pooled, lyophilized, redissolved in 50 mM Hepes(pH 7.5), 0.1 M NaCl, 2 mM CaCl2, and 0.1% triton x-100, and stored at−20° C. until needed. The concentration of recombinant bikunin wasdetermined by amino acid analysis.

Results. Recombinant bikunin was purified from baculovirus cell culturesupernatant using a 2-step purification protocol as shown below, toyield an active trypsin inhibitor (Table 8 below). TABLE 8 Purificationof recombinant bikunin from transformed culture supernatant TABLE 8Specific Purification Vol OD 280 Units activity Step (ml) OD 280/mltotal (U) (U/OD) Supernatant 2300.0 9.0 20,700 6,150,000 297 Kallikrein23.0 0.12 2.76 40,700 14,746 affinity C18 reverse- 0.4 3.84 1.54 11,11172,150 phase

Chromatography of the crude material over an immobilized bovinepancreatic kallikrein affinity column selectively isolated 0.013% of theprotein and 0.67% of the trypsin inhibitory activity present. Themajority of the trypsin inhibitory activity present in the startingsupernatant did not bind to the immobilized kallikrein and is notrelated to bikunin (results not shown). Subsequent chromatography usingC18 reverse-phase yielded a further purification of 5-fold, with arecovery of 0.2%. The final preparation was highly pure by SDS-PAGE(FIG. 13), exhibiting an Mr of 21.3 kDa, and reacted on immunoblots torabbit anti-placental bikunin 102-159 (not shown). N-terminal sequencing(26 cycles) yielded the expected sequence for mature placental bikunin(FIG. 4F) starting at residue +1 (ADRER . . . ), showing that the signalpeptide was correctly processed in Sf9 cells.

Purified placental bikunin from Sf9 cells (100 pmol) waspyridylethyl-alkylated, CNBr digested and then sequenced withoutresolution of the resulting fragments. Sequencing for 20 cycles yieldedthe following N-terminii: Sequence Amount Placental bikunin residue #LRCFrQQENPP-PLG----- 21 pmol 154-168 (SEQ ID NO: 63)ADRERSIHDFCLVSKVVGRC 20 pmol   1-20 (SEQ ID NO: 64) FNYeEYCTANAVTGPCRASF16 pmol 100-119 (SEQ ID NO: 65) Pr--Y-V-dGS-Q-F-Y-G  6 pmol  25-43 (SEQID NO: 66)Thus N-terminii corresponding to each of the expected four fragmentswere recovered. This confirms that the Sf9 expressed protein containedthe entire ectodomain sequence of placental bikunin (1-170). N-terminalsequencing (50 cycles) of an additional sample of undigested PlacentalBikunin (1-170) resulted in an amino acid sequence which at cycle 30 wasdevoid of any PTH-amino acid (PTH-asparagine was expected). A similarresult was obtained upon sequencing of the natural protein from humanplacenta (Example 7) and is consistent with this residue beingglycosylated as predicted from the amino acid sequence surrounding thisasparagine residue. Furthermore, the cysteine residues within thisregion were also silent consistent with their participation in disulfidebonding.

EXAMPLE 10

Inhibition Specificity of Purified Placental Bikunin Derived from Sf9Cells.

The in vitro specificity of recombinant bikunin was determined using thematerials and methods as described in Examples 3, 4 and 7. In addition,the inhibition of human tissue kallikrein by bikunin was measured by theincubation of 0.35 nM human tissue kallikrein recombinant bikunin inbuffer containing 50 mM Tris (pH 9.0), 50 mM NaCl, and 0.01% tritonx-100. After 5 min. at 37° C., 5 μl of 2 mM PFR-AMC was added and thechange in fluorescence monitored.

Inhibition of tissue plasminogen activator (tPA) was also determined asfollows: tPA (single chain form from human melanoma cell culture fromSigma Chemical Co, St Louis, Mo.) was pre-incubated with inhibitor for 2hr at room temperature in 20 mM Tris buffer pH 7.2 containing 150 mMNaCl, and 0.02% sodium azide. Reactions were subsequently initiated bytransfer to a reaction system comprising the following initial componentconcentrations: tPA (7.5 nM), inhibitor 0 to 6.6 μM,DIle-Lpro-Larg-pNitroaniline (1 mM) in 28 mM Tris buffer pH 8.5containing 0.004% (v/v) triton x-100 and 0.005% (v/v) sodium azide.Formation of p-Nitroaniline was determined from the A405 nm measuredfollowing incubation at 37 C for 2 hr.

The table below show the efficacy of recombinant bikunin as an inhibitorof various serine proteases in vitro. Data is shown compared againstdata obtained for screening inhibition using either recombinant bikunin,or aprotinin. TABLE 9 Comparisons of Ki values for the inhibition ofvarious proteases by recombinant placental bikunin (1-170) or aprotininTABLE 9 Protease Recombinant Bikunin Aprotinin (concentration) Ki (nM)Ki(nM) Trypsin (48.5 pM) 0.064 0.8 Human Plasma 0.18 19.0 Kallikrein(2.5 nM) Human Tissue 0.04 0.004 Kallikrein (0.35 nM) Bovine Pancreatic0.12 0.02 Kallikrein (100 pM) Human Plasmin (50 pM) 0.23 1.3 factor Xa(0.87 nM) 180 5% Inhibition at 31 μM factor XIa (0.1 nM) 3.0 288 tissueplasminogen <60 no inhibition activator (7.5 nM) at 6.6 μM Tissue FactorVila 800 no inhibition at 1 μM

The results show that recombinant bikunin can be expressed in insectcells to yield an active protease inhibitor that is effective against atleast five different serine protease inhibitors. Recombinant bikunin wasmore potent than aprotinin against human plasma kallikrein, trypsin andplasmin. Surprisingly, the recombinant bikunin was more potent that thesynthetically derived bikunin fragments (7-64) and (102-159) against allenzymes tested. These data show that recombinant bikunin is moreeffective than aprotinin, using in vitro assays, and that one wouldexpect better in vivo potency.

Besides measuring the potencies against specific proteases, the capacityof placental bikunin (1-170) to prolong the activated partialthromboplastin time (APTT) was evaluated and compared with the activityassociated with aprotinin. Inhibitor was diluted in 20 mM Tris buffer pH7.2 containing 150 mM NaCl and 0.02% sodium azide and added (0.1 ml) toa cuvette contained within an MLA Electra^(R) 800 Automatic CoagulationTimer coagulometer (Medical Laboratory Automation, Inc., Pleasantville,N.Y.). The instrument was set to APTT mode with a 300 sec. activationtime and the duplicate mode. Following addition of 0.1 ml of plasma(Specialty Assayed Reference Plasma lot 1-6-5185, Helena Laboratories,Beaumont, Tex.), the APTT reagent (Automated APTT-lot 102345, fromOrganon Teknika Corp., Durhan, N.C.) and 25 mM CaC12 were automaticallydispensed to initiate clotting, and the clotting time was monitoredautomatically. The results (FIG. 14) showed that a doubling of theclotting time required approximately 2 μM final aprotinin, but only 0.3μM Sf9 derived placental bikunin. These data show that placental bikuninis an effective anticoagulant, and usefull as a medicament for diseasesinvolving pathologic activation of the intrinsic pathway of coagulation.

Although certain embodiments of the invention have been described indetail for the purpose of illustration, it will be readily apparent tothose skilled in the art that the methods and formulations describedherein may be modified without departing from the spirit and scope ofthe invention. Accordingly, the invention is not limited except as bythe appended claims.

1. A substantially purified protein, having serine protease inhibitoryactivity, selected from the group of proteins consisting of materialseach of which comprises one of the following amino acid sequences, theamino acids of said sequences being numbered in accordance with theamino acid sequence of native human placental bikunin shown in FIG. 4Fin which the N-terminal residue generated by removal of signal peptideis designated as residue 1: (SEQ ID NO.:52) ADRERSIHDF CLVSKWGRCRASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50 YLTKEECLKK CATVTENATG DLATSRNAADSSVPSAPRRQ DSEDHSSDMF 100 NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCRGNKNSYRSEE 150 ACMLRCFRQQ ENPPLPLGSK; (SEQ ID NO.: 49) MAQLCGLRRSRAFLALL GSLLLSGVLA −1 ADRERSIHDF CLVSKWGRC RASMPRWWYN VTDGSCQLFVYGGCDGNSNN 50 YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150 ACMLRCFRQQENPPLPLGSK WVLAGLFVM VLILFLGASM VYLIRVARRN 200 QERALRTVWS SGDDKEQLVKNTYVL. 225 (SEQ ID NO.: 71) ADRERSIHDF CLVSKWGRC RASMPRWWYN VTDGSCQLFVYGGCDGNSNN 50 YLTKEECLKK VATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150 ACMLRCFRQQENPPLPLGSK WVLAGLFVM VLILFLGASM VYLIRVARRN 200 QERALRTVWS SGDDKEQLVKNTYVL, 225 (SEQ ID NO.: 2) AGSFLAWL GSLLLSGVLA −1 ADRERSIHDF CLVSKWGRCRASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50 YLTKEECLKK CATVTENATG DLATSRNAADSSVPSAPRRQ DSEDHSSDMF 100 NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCRGNKNSYRSEE 150 ACMLRCFRQQ ENPPLPLGSK VWLAGAVS, 179 (SEQ ID NO.: 45) MLRAEADGVSRLL GSLLLSGVLA −1 ADRERSIHDF CLVSKWGRC RASMPRWWYN VTDGSCQLFVYGGCDGNSNN 50 YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100NYEEYCTANA VTGPCRASFP TWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150 ACMLRCFRQQENPPLPLGSK WVLAGLFVM VLILFLGASM VYLIRVARRN 200 QERALRTVWS SGDDKEQLVKNTYVL, 225 (SEQ ID NO.: 47) MAQLCGL RRSRAFLALL GSLLLSGVLA −1 ADRERSIHDFCLVSKWGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50 YLTKEECLKK CATVTENATGDLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100 NYEEYCTANA VTGPCRASFP RWYFDVERNSCNNFIYGGCR GNKNSYRSEE 150 ACMLRCFRQQ ENPPLPLGSK WVLAGLFVM VLILFLGASMVYLIRVARRN 200 QERALRTVWS FGD, 213 (SEQ ID NO.: 70) ADRERSIHDF CLVSKWGRCRASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50 YLTKEECLKK CATVTENATG DLATSRNAADSSVPSAPRRQ DSEDHSSDMF 100 NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCRGNKNSYRSEE 150 ACMLRCFRQQ ENPPLPLGSK WVLAGLFVM VLILFLGASM VYLIRVARRN 200QERALRTVWS FGD; 213 (SEQ ID NO.: 4) IHDF CLVSKWGRC RASMPRWWYN VTDGSCQLFVYGGCDGNSNN 50 YLTKEECLKK CATV; 64 (SEQ ID NO.: 5) CLVSKWGRC RASMPRWWYNVTDGSCQLFV YGGCDGNSNN 50 YLTKEECLKK C; 61 (SEQ ID NO.: 6) YEEYCTANAVTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150 ACMLRCFRQ; 159 (SEQ IDNO.: 7) CTANAVTGPC RASFPRWYFD VERNSCNNFI YGGCRGNKNS YRSEE 150 ACMLRC;156 (SEQ ID NO.: 3) IHDF CLVSKWGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 75 NYEEYCTANAVTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 125 ACMLRCFRQ; 159 (SEQ IDNO.: 50) CLVSKWGRC RASMPRWWYN VTDGSCQLFV YGGDCGNSNN 50 YLTKEECLKKCATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100 NYEEYCTANA VTGPCRASFPRWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150 AGMLRC; 156 (SEQ ID NO.: 1)ADRERSIHDF CLVSKWGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 25 YLTKEECLKKCATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 75 NYEEYCTANA VTGPCRASFPRWYFDVERNS CNNFIYGGCR GNKNSYRSEE 125 ACMLRCFRQQ ENPPLPLGSK VWLAGAVS; 179and (SEQ ID NO.: 8) ADRERSIHDF CLVSKWGRC RASMPRWWYN VTDGSCQLFVYGGCDGNSNN 50 YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DS. 92


2. A protein as in claim 1, wherein said protein is glycosylated, orcontains at least one intra-chain cysteine-cysteine disulfide bond, oris both glycosylated and contains at least one intra-chaincysteine-cysteine disulfide bond.
 3. A pharmaceutical composition forinhibiting serine protease activity, comprising a protein of claim 1plus a pharmaceutically acceptable carrier.
 4. An isolated nucleic acidsequence which encodes for a protein of claim
 1. 5. A self-replicatingprotein expression vector containing a nucleic acid sequence whichencodes for and is capable of expressing a protein of claim
 1. 6. Amethod for inhibiting serine protease activity comprising contactingserine protease with an effective amount of at least one protein ofclaim
 1. 7. A method for treating a condition of brain edema, spinalcord edema, multiple sclerosis, ischemia, perioperative blood loss,sepsis, septic shock, fibrosis, disease associated with pathologic bloodcoagulation or clotting, polytrauma, stroke, cerebral or subarachnoidhemorrhage, inflammation of the brain, inflammation of the spinal cord,cerebral infection, cerebral granulomatosis, spinal infection, spinalgranulomatosis, open heart surgery, gastric cancer, cervical cancer, orprevention of metastasis comprising administering to a subject havingsuch a condition and effective amount of the protein of claim 1 to asubject who requires treatment.
 8. The method of claim 7 wherein saidcondition is brain edema, spinal cord edema, multiple sclerosis,ischemia, perioperative blood loss, sepsis, septic shock, fibrosis,disease associated with pathologic blood coagulation or clotting,stroke, cerebral or subarachnoid hemorrhage, inflammation of the brain,inflammation of the spinal cord, cerebral infection, cerebralgranulomatosis, spinal infection, spinal granulomatosis, or open heartsurgery.
 9. The method of claim 7 wherein said condition is gastriccancer, cervical cancer, or prevention of metastasis.
 10. A method forthe preparation of a medicament for the treatment of brain edema, spinalcord edema, multiple sclerosis, ischemia, perioperative blood loss,sepsis, septic shock, fibrosis, disease associated with pathologic bloodcoagulation or clotting, stroke, cerebral or subarachnoid hemorrhage,inflammation of the brain, inflammation of the spinal cord, cerebralinfection, cerebral granulomatosis, spinal infection, spinalgranulomatosis, open heart surgery, gastric cancer cervical cancer, orprevention of metastasis, comprising combining an effective amount of aprotein of claim 1 with a suitable pharmaceutical carrier or excipient.11. A method for preparing a protein of claim 1 using recombinant DNAtechnology.